AU694232B2 - Receptor for oncostatin M - Google Patents

Abstract

A novel polypeptide functions as the beta chain of an oncostatin M receptor and is thus designated OSM-Rbeta. Heterodimeric receptor proteins comprising OSM-Rbeta and gp130 bind oncostatin M and find use in inhibiting biological activities mediated by oncostatin M.

Description

1

TITLE

Receptor for Oncostatin M BACKGROUND OF THE INVENTION Oncostatin M is a secreted single-chain polypeptide cytokine that regulates the growth of certain tumor-derived and normal cell lines. A number of cell types have been found to bind the oncostatin M protein. See, for example, Linsley et al., J. Biol. Chem., 264:4282 (1989). Oncostatin M has been shown to inhibit proliferation of a number of tumor cell types (Linsley et al. supra). In contrast, however, this protein has been implicated in stimulating proliferation of Kaposi's sarcoma cells (Nair et al., Science 255:1430, 1992; Miles et al., Science 255:1432, 1992; and Cai et al., Am. J. Pathol. 145:74, 1994).

Identifying and isolating oncostatin M-binding proteins, such as cell surface oncostatin M receptors, is desirable for such reasons as enabling study of the biological signal transduced via the receptor. Such receptors in soluble form also could be used to competitively inhibit a biological activity of oncostatin M in various in vitro assays or in vivo procedures. A soluble form of the receptor could 20 be administered to bind oncostatin M in vivo, thus inhibiting the binding of oncostatin M to endogenous cell surface receptors, for example.

o0 A protein known as gp130 has been found to bind oncostatin M, but with relatively low affinity (Gearing et al., Science 255:1434,1992). Heterodimeric receptors comprising a leukemia inhibitory factor (LIF) receptor and gp130 bind 25 oncostatin M with higher affinity than does gp130 alone, but also bind LIF with high affinity (Gearing et al., supra). For certain applications, a receptor that binds oncostatin M with high affinity, but that does not function as a high affinity LIF receptor, would be advantageous. Prior to the present invention, no such receptor had been identified or isolated.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

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11-~11~ WO 95/33059 PCT/US95/06530 SUMMARY OF THE INVENTION The present invention provides a novel polypeptide that is designated herein as the oncostatin M receptor B subunit (OSM-RB). Also provided is a receptor comprising OSM- RB linked (preferably covalently) to an oncostatin M-binding protein known as gp130. The gpl30 polypeptide may be covalently linked to the OSM-RB polypeptide by any suitable means, such as via a cross-linking reagent or a polypeptide linker. In one embodiment of the invention, the receptor is a fusion protein produced by recombinant DNA technology.

This receptor comprising OSM-RB and gpl30 binds oncostatin M at levels greater than does gpl30 alone. Disorders mediated by oncostatin M may be treated by administering a therapeutically effective amount of this inventive receptor to a patient afflicted with such a disorder.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents a Scatchard analysis generated from an assay for binding of radioiodinated oncostatin M by cells expressing recombinant gpl30. The assay is described in example 2.

Figure 2 presents a Scatchard analysis of the results of an assay for binding of radioiodinated oncostatin M by cells expressing both recombinant gpl30 and recombinant OSM-RB. As described in example 2, the data in figure 2 demonstrate higher affinity oncostatin M binding compared to the oncostatin M binding by gpl30 alone depicted in figure 1.

Figure 3 is a bar graph representing binding of leukemia inhibitory factor (LIF) and oncostatin M to various receptor proteins, as described in example DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel polypeptide designated the oncostatin M receptor B subunit (OSM-RB). Isolated DNA encoding OSM-RB, expression vectors containing OSM-RB DNA, and host cells transformed with such expression vectors are disclosed. Methods for production of recombinant OSM-RB polypeptides, including soluble forms of the protein, are also disclosed. Antibodies immunoreactive with the novel polypeptide are provided herein as well.

Another embodiment of the invention is directed to a receptor capable of binding oncostatin M, wherein the receptor comprises OSM-RB and gpl30. The receptor finds use in various in vitro and in vivo procedures, including treatment of disorders mediated by oncostatin M.

DNA and encoded amino acid sequences of the OSM-RB cDNA isolated in example 1 are presented in SEQ ID NO:5 and SEQ ID NO:6. The encoded protein comprises (from N- to C-terminus) a signal peptide (amino acids -27 to -1 of SEQ ID NO:6) followed by an .i WO 95/33059 PCT/US95/06530 extracellular domain (amino acids 1 to 714), a transmembrane region (amino acids 715 to 734) and a cytoplasmic domain (amino acids 735 to 952). E. coli cells transformed with a recombinant vector comprising OSM-RB cDNA in the cloning vector pBluescript® SKwere deposited with the American Type Culture Collection, Rockville, MD, on August 16, 1994, and assigned accession no. ATCC 69675.

The binding assay described in example 2 compared the binding of oncostatin M by cells expressing either gpl30 alone or both gpl30 and OSM-RB. The cells expressing both and OSM-RB exhibited higher affinity oncostatin M binding than did cells expressing gpl30 alone. The assay described in example 5 demonstrates that OSM-RB alone does not bind oncostatin M at a detectable level. However, proteins expressed by cells co-transfected with both a soluble OSM-RB/Fc fusion protein-encoding vector and a soluble gpl30/Fc fusion protein-encoding vector bound oncostatin M at higher levels than did proteins expressed by cells transfected with a soluble gpl30/Fc-encoding vector alone.

In one embodiment, a receptor of the present invention comprises gpl30 covalently linked to OSM-RB by any suitable means, such as via a cross-linking reagent or a polypeptide linker. The gpl30 and OSM-RB proteins are covalently linked in a manner that does not interfere with the resulting receptor's ability to bind oncostatin M. In one embodiment, the receptor is a fusion protein produced by recombinant DNA technology.

Alternatively, the receptor may comprise gpl30 non-covalently complexed with OSM-RB. Non-covalent bonding of gpl30 to OSM-RB may be achieved by any suitable means that does not interfere with the receptor's ability to bind oncostatin M. In one approach, a first compound is attached to OSM-RB and a second compound that will noncovalently bond to the first compound is attached to gpl30. Examples of such compounds are biotin and avidin. The receptor is thus formed through the non-covalent interactions of biotin with avidin. In one embodiment of the invention, OSM-RB and gpl30 are recombinant polypeptides, each purified from recombinant cells and then non-covalently bonded together to form the receptor. A host cell may be transformed with two different expression vectors such that both OSM-RB and gpl30 are produced by the recombinant host cell. OSM-RB and gpl30 produced by such transformed host cells may associate to form a complex through non-covalent interactions. When such transformed cells express the membrane-bound forms of the proteins, such cells are useful in various assays, including competition assays.

The protein designated gpl30 herein has been purified from cellular sources that include placental tissue and a myeloma cell line U266. A number of additional cell types have been found to express gpl30 mRNA, as reported by Hibi et al., in Cell 63:1149 (1990). gpl30 has been reported to be involved in the formation of high affinity interleukin-6 binding sites and in IL-6 signal transduction (Hibi et al. supra). gpl30 also serves as an affinity converter for the LIF receptor (Gearing et al., Science 255:1434, I -CilO~ i-i i _I WO 95/33059 PCT/US95/06530 1992). The cloning and expression of cDNA encoding a full length gpl30 protein has been reported by Hibi et al., supra, which is hereby incorporated by reference in its entirety.

As used herein, the terms OSM-RB and gpl30 include variants and truncated forms of the native proteins that possess the desired biological activity. Variants produced by adding, substituting, or deleting amino acid(s) in the native sequence are discussed in more detail below.

One example of an OSM-RB polypeptide is that encoded by the cDNA clone described in example 1 encoded by the OSM-Rp cDNA insert of the recombinant vector in deposited strain ATCC 69675). Other OSM-RB polypeptides include those lacking all or part of the transmembrane region or the cytoplasmic domain of the protein.

1 Additional truncated OSM-RB polypeptides may be chosen with regard to sequences that i are conserved in the hematopoietin receptor family. The desirability of including the signal sequence depends on such factors as the position of the OSM-RB in a fusion protein and 1 the intended host cells when the receptor is to be produced via recombinant DNA technology.

One example of a suitable gpl30 polypeptide is that comprising the amino acid sequence presented in SEQ ID NO:2. E. coli strain DH5x cells transformed with a encoding recombinant vector designated B10G/pDC303 were deposited with the American i Type Culture Collection, Rockville, Maryland, on November 14, 1991, and assigned ATCC accession number 68827. The mammalian expression vector pDC303 (into which the gpl30 cDNA has been inserted to form B10G/pDC303) is also known as SF CAV, and has been described in PCT application WO 93/19777. The nucleotide sequence of the cDNA contained in plasmid B10G/pDC303 and the amino acid sequence encoded thereby are presented in SEQ ID NO:1 and SEQ ID NO:2. The protein comprises (in order from the N-terminus to the C-terminus) a 22-amino acid signal sequence, complete extracellular domain (amino acids 1-597), a transmembrane region (beginning with amino acid 598), and a partial cytoplasmic domain (amino acids 621-686).

Alternatively, the gp130 protein disclosed by Hibi et al. supra may be employed.

The eighth amino acid of the signal peptide is valine in the sequence reported by Hibi et al., but is leucine in SEQ ID NO:2 (at position This difference in amino acid sequence may be attributable to genetic polymorphism (allelic variation among individuals producing the protein). In addition, the gpl30 protein of SEQ ID NO:2 is truncated within the cytoplasmic domain, terminating with the leucine residue found at position 708 in the sequence presented in Hibi et al. supra. Although truncated, the gpl30 protein of SEQ ID NO:2 comprises the extracellular domain responsible for oncostatin M binding, and thus is suitable for use as a component of the receptors of the present invention.

Regions of the gpl30 protein corresponding to domains that are conserved among certain receptors are discussed by Hibi et al, supra, at page 1150, column 2, and page WO 95/33059 PCTUS95/06530 1151, column 1. Other truncated gp130 polypeptides chosen to include these conserved regions may be employed.

Soluble OSM-RB and gpl30 polypeptides are preferred for certain applications. In one embodiment of the present invention, the receptor comprises soluble OSM-RB covalently attached to soluble gpl30. "Soluble OSM-RB" as used in the context of the present invention refers to polypeptides that are substantially similar in amino acid sequence to all or part of the extracellular region of a native OSM-RB and that, due to the lack of a transmembrane region that would cause retention of the polypeptide on a cell membrane, are secreted upon expression. Suitable soluble OSM-RB polypeptides retain the desired biological activity. Soluble OSM-RB may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble OSM-RB protein is capable of being secreted.

Likewise, the term "soluble gpl30" as used herein refers to proteins that are substantially similar in amino acid sequence to all or part of the extracellular region of a native gpl30 and are secreted upon expression but retain the desired biological activity.

Soluble gpl30 may include part of the transmembrane region, cytoplasmic domain, or other sequences, as long as the polypeptide is secreted.

In one embodiment, soluble OSM-RB and gpl30 polypeptides include the entire extracellular domain. To effect secretion, the soluble polypeptides comprise the native signal peptide or a heterologous signal peptide. Thus, examples of soluble OSM-RB polypeptides comprise amino acids -27 to 714 or 1 to 714 of SEQ ID NO:6. Examples of soluble gpl30 polypeptides comprise amino acids -22 to 597 or 1 to 597 of SEQ ID NO:2.

Additional examples of soluble gpl30 polypeptides are those lacking from one to all three of the fibronectin domains found within the extracellular domain, as described in example 4 below. These soluble gp130 polypeptides include those comprising amino acids -22 to y or 1 to y of SEQ ID NO:2, wherein y is an integer between 308 and 597, inclusive.

A soluble fusion protein comprising amino acids -27 through 432 of the OSM-Rp of SEQ ID NO:6 fused to an antibody Fc region polypeptide is described in example The OSM-Rp moiety of the fusion protein, which is a fragment of the OSM-Rp extracellular domain, retained the desired biological activity. Thus, examples of soluble OSM-Rp polypeptides comprise amino acids -27 to x, or 1 to x of SEQ ID NO:6, wherein x is an integer between 432 and 714, inclusive.

Soluble OSM-RB and soluble gpl30 may be identified (and distinguished from their non-soluble membrane-bound counterparts) by separating intact cells which express the desired protein from the culture medium, by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein. The culture medium may be assayed using procedures which are similar or identical to those described in the examples below.

-I i i -i L~YI WO 95/33059 PCT/US95/06530 The presence of OSM-RB or gpl30 in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the desired protein. Soluble OSM-RB and soluble gpl30 may be naturally-occurring forms of these proteins. Alternatively, soluble fragments of OSM-RB and gpl30 proteins may be produced by recombinant DNA technology or otherwise isolated, as described below.

The use of soluble forms of OSM-RB and gpl30 is advantageous for certain applications. Purification of the proteins from recombinant host cells is facilitated, since the soluble proteins are secreted from the cells. Further, a receptor of the present invention comprising soluble OSM-RB and gpl30 proteins is generally more suitable for intravenous administration.

With respect to the foregoing discussion of signal peptides and the various domains of the gpl30 and OSM-RB proteins, the skilled artisan will recognize that the abovedescribed boundaries of such regions of the proteins are approximate. For example, although computer programs that predict the site of cleavage of a signal peptide are available, cleavage can occur at sites other than those predicted. Further, it is recognized that a protein preparation can comprise a mixture of protein molecules having different Nterminal amino acids, due to cleavage of the signal peptide at more than one site. In addition, the OSM-Rp transmembrane region was identified by computer program prediction in combination with homology to the transmembrane region of the LIF receptor protein described by Gearing et al. (EMBO J. 10:2839, 1991). Thus, soluble OSM-RB polypeptides comprising the extracellular domain include those having a C-terminal amino acid that may vary from that identified above as the C-terminus of the extracellular domain.

Further, post-translational processing that can vary according to the particular expression system employed may yield proteins having differing N-termini. Such variants that retain the desired biological activities are encompassed by the terms "OSM-RB polypeptides" and polypeptides" as used herein.

Truncated OSM-RB and gpl30, including soluble polypeptides, may be prepared by any of a number of conventional techniques. In the case of recombinant proteins, a DNA fragment encoding a desired fragment may be subcloned into an expression vector.

Alternatively, a desired DNA sequence may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels.

Linkers containing restriction endonuclease cleavage site(s) may be employed to insert the desired DNA fragment into an expression vector, or the fragment may be digested at cleavage sites naturally present therein. Oligonucleotides that reconstruct the N- or Cterminus of a DNA fragment to a desired point may be synthesized. The oligonucleotide may contain a restriction endonuclease cleavage site upstream of the desired coding sequence and position an initiation codon (ATG) at the N-terminus of the coding sequence.

C i c t- I--I-a IC WO 95/33059 PCTIUS95/06530 The well known polymerase chain reaction procedure also may be employed to isolate a DNA sequence encoding a desired protein fragment. Oligonucleotide primers comprising the desired termini of the fragment are employed in such a polymerase chain reaction. Any suitable PCR procedure may be employed. One such procedure is described in Saiki et al., Science 239:487 (1988). Another is described in Recombinant DNA Methodology, Wu et al., eds., Academic Press Inc., San Diego (1989), pp. 189-196. In general, PCR reactions involve combining the 5' and 3' oligonucleotide primers with template DNA (in this case, OSM-RB or gpl30 DNA) and each of the four deoxynucleoside triphosphates, in a suitable buffered solution. The solution is heated, from 95" to 100°C) to denature the double-stranded DNA template and is then cooled before addition of a DNA polymerase enzyme. Multiple cycles of the reactions are carried out in order to amplify the desired DNA fragment.

The gpl30 polypeptide is attached to the OSM-RB polypeptide through a covalent or non-covalent linkage. Covalent attachment is preferred for certain applications, e.g. in vivo use, in view of the enhanced stability generally conferred by covalent, as opposed to non-covalent, bonds. In constructing the receptor of the present invention, covalent linkage may be accomplished via cross-linking reagents, peptide linkers, or any other suitable technique.

Numerous reagents useful for cross-linking one protein molecule to another are known. Heterobifunctional and homobifunctional linkers are available for this purpose from Pierce Chemical Company, Rockford, Illinois, for example. Such linkers contain two functional groups esters and/or maleimides) that will react with certain functional groups on amino acid side chains, thus linking one polypeptide to another.

One type of peptide linker that may be employed in the present invention separates gpl30 and OSM-RB domains by a distance sufficient to ensure that each domain properly folds into the secondary and tertiary structures necessary for the desired biological activity.

The linker also should allow the extracellular domains of gpl30 and OSM-Rp to assume the proper spatial orientation to form the binding site for oncostatin M.

Suitable peptide linkers are known in the art, and may be employed according to conventional techniques. Among the suitable peptide linkers are those described in U.S.

Patents 4,751,180 and 4,935,233, which are hereby incorporated by reference. A peptide linker may be attached to gpl30 and to OSM-R by any of the conventional procedures used to attach one polypeptide to another. The cross-linking reagents available from Pierce Chemical Company as described above are among those that may be employed. Amino acids having side chains reactive with such reagents may be included in the peptide linker, at the termini thereof. Preferably, a fusion protein comprising gpl30 joined to OSM- RB via a peptide linker is prepared by recombinant DNA technology.

I i I I c~e L-T--~i~ras~a ij~ii ~q _1 WO 95/33059 PCT/US95/06530 In one embodiment of the invention, OSM-RB and gpl30 are linked via polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, by Ashkenazi et al. (PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990). As one example, a polypeptide derived from the Fc region of an antibody may be attached to the C-terminus of OSM-RB.

A separate Fc polypeptide is attached to the C-terminus of gpl30. Disulfide bonds form between the two Fc polypeptides in the so-called hinge region, where interchain disulfide bonds are normally present in antibody molecules), producing a heterodimer comprising the pl30 an-d to OSM-RB/Fc fusion protein linked to the gpl30/Fc fusion protein. Advantageously, host cells are co-transfected with two different expression vectors, one encoding soluble OSM-RB/Fc and the other encoding soluble gpl30/Fc. The heterodimer is believed to form intracellularly or during secretion.

The term "Fc polypeptide" as used herein includes native and mutein forms, as well as truncated i polypeptides containing the hinge region that promotes dimerization.

cDNA encoding a single chain polypeptide derived from the Fc region of a human IgG1 antibody has been cloned into the pBluescript SK® cloning vector (Stratagene Cloning Systems, LaJolla, CA) to produce a recombinant vector designated hIgG1Fc. A unique BglII site is positioned near the 5' end of the inserted Fc encoding sequence. An Spel site is immediately downstream of the stop codon. The DNA and encoded amino acid sequences of the cloned Fc cDNA are presented in SEQ ID NO:3 and SEQ ID NO:4. The Fc polypeptide encoded by the cDNA extends from the N-terminal hinge region to the native C-terminus, is an essentially full-length antibody Fc region. One suitable mutein of this Fc polypeptide is described in U.S. patent application serial no. 08/097,827, hereby incorporated by reference. The mutein exhibits reduced affinity for Fc receptors.

Homodimers comprising two OSM-RB/Fc polypeptides or two polypeptides linked via disulfide bonds are also produced by certain of the transfected host cells disclosed herein. The homodimers may be separated from each other and from the heterodimer by virtue of differences in size by gel electrophoresis). The heterodimer also may be purified by sequential immunoaffinity chromatography (described below).

In an alternative embodiment, a first fusion polypeptide comprising gpl30 (or a fragment thereof) upstream of the constant region of an antibody light chain (or a fragment thereof) is prepared. A second fusion polypeptide comprises OSM-RB upstream of the constant region of an antibody heavy chain (or a heavy chain fragment, the N-terminus of which extends at least through the CH 1 region. Disulfide bond(s) form between the light chain fusion polypeptide and the OSM-RB-heavy chain fusion polypeptide, thus producing a receptor of the present invention. As a further alternative, an OSM-R3antibody light chain fusion polypeptide is prepared and combined with (disulfide bonded 8 re I 1

I

WO 95/33059 PCT/US95/06530 to) a fusion polypeptide comprising gp130 fused to an antibody heavy chain. When two of the foregoing disulfide bonded molecules are combined, additional disulfide bonds form between the two Fc regions. The resulting receptor of the present invention comprising four fusion polypeptides resembles an antibody in structure and displays the oncostatin M binding site bivalently.

The gpl30 and OSM-RB polypeptides may be separately purified from cellular sources, and then linked together. Alternatively, the receptor of the present invention may i be produced using recombinant DNA technology. The gpl30 and OSM-RB polypeptides may be produo- d separately and purified from transformed host cells for subsequent covalent linkage. In one embodiment of the present invention, a host cell is transformed/transfected with foreign DNA that encodes gpl30 and OSM-RB as separate i polypeptides. The two polypeptides may be encoded by the same expression vector with start and stop codons for each of the two genes, or the recombinant cells may be cotransfected with two separate expression vectors. In another embodiment, the receptor is produced as a fusion protein in recombinant cells.

In one embodiment of the present invention, the receptor protein is a recombinant fusion protein of the formula: RI-L-R2 or R 2

-L-RI

wherein R 1 represents gpl30 or a gpl30 fragment; R 2 represents OSM-RB or an OSM-RB fragment; and L represents a peptide linker.

The fusion proteins of the present invention include constructs in which the Cterminal portion of gpl30 is fused to the linker which is fused to the N-terminal portion of OSM-RB, and also constructs in which the C-terminal portion of OSM-R8 is fused to the linker which is fused to the N-terminal portion of gpl30. gpl30 is covalently linked to OSM-RB in such a manner as to produce a single protein which retains the desired biological activities of gpl30 and OSM-RB. The components of the fusion protein are listed in their order of occurrence the N-terminal polypeptide is listed first, followed by the linker and then the C-terminal polypeptide).

A DNA sequence encoding a fusion protein is constructed using recombinant DNA techniques to insert separate DNA fragments encoding gpl30 and OSM-RB into an appropriate expression vector. The 3' end of a DNA fragment encoding gpl30 is ligated (via the linker) to the 5' end of the DNA fragment encoding OSM-RB with the reading frames of the sequences in phase to permit translation of the mRNA into a single biologically active fusion protein. Alternatively, the 3' end of a DNA fragment encoding OSM-RB may be ligated (via the linker) to the 5' end of the DNA fragment encoding with the reading frames of the sequences in phase to permit translation of the mRNA into a single biologically active fusion protein. A DNA sequence encoding an Nll(llt i Li WO 95/33059 PCT/US95/06530 terminal signal sequence may be retained on the DNA sequence encoding the N-terminal polypeptide, while stop codons, which would prevent read-through to the second (Cterminal) DNA sequence, are eliminated. Conversely, a stop codon required to end translation is retained on the second DNA sequence. DNA encoding a signal sequence is preferably removed from the DNA sequence encoding the C-terminal polypeptide.

A DNA sequence encoding a desired polypeptide linker may be inserted between, and in the same reading frame as, the DNA sequences encoding gpl30 and OSM-RB using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding the linker and containing appropriate restriction endonuclease cleavage sites may be ligated between the sequences encoding gpl30 and OSM-RB.

Alternatively, a chemically synthesized DNA sequence may contain a sequence complementary to the 3' terminus (without the stop codon) of either gpl30 or OSM-RB, followed by a linker-encoding sequence which is followed by a sequence complementary to the 5' terminus of the other of gpl30 and OSM-RB. Oligonucleotide directed mutagenesis is then employed to insert the linker-encoding sequence into a vector containing a direct fusion of gpl30 and OSM-RB.

The present inveption provides isolated DNA sequences encoding the abovedescribed fusion proteins comprising gpl30, OSM-RB, and a peptide linker. DNA encoding the novel OSM-RB polypeptides disclosed herein is also provided, as is DNA encoding OSM-RB polypeptides fused to immunoglobin-derived polypeptides. OSM-RBencoding DNA encompassed by the present invention includes, for example, cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. Genomic OSM-RB DNA may be isolated using the cDNA isolated in Example 1, or fragments thereof, as a probe using standard techniques.

Also provided herein are recombinant expression vectors containing the isolate,) DNA sequences. "Expression vector" refers to a replicable DNA construct used to express DNA which encodes the desired protein and which includes a transcriptional unit comprising an assembly of genetic element(s) having a regulatory role in gene expression, for example, promoters, operators, or enhancers, operatively linked to a DNA sequence encoding a desired protein which is transcribed into mRNA and translated into protein, and appropriate transcription and translation initiation and termination sequences. The choice of promoter and other regulatory elements generally varies according to the intended host cell.

In the expression vectors, regulatory elements controlling transcription or translation are generally derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from retroviruses also may be employed.

I

WO 95/33059 PCT/US95/06530 DNA regions are operably linked when they are functionally related to each other.

For example, DNA encoding a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if the polypeptide is expressed as a precursor that is secreted through the host cell membrane; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, "operably linked" means contiguous and, in the case of secretory leaders, contiguous and in reading frame.

Transformed host cells are cells which have been transformed or transfected with foreign DNA using recombinant DNA techniques. In the context of the present invention, the foreign DNA includes a sequence encoding the inventive proteins. Host cells may be transformed for purposes of cloning or amplifying the foreign DNA, or may be transformed with an expression vector for production of the protein. Suitable host cells include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant disclosure of which is hereby incorporated by reference.

Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host. Examples of suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and this provides simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectors include the 1-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and tac promoter 14. An isolated DNA encoding an OSM-Rp polypeptide, wherein said DNA is selected from the group consisting of: /2 WO 95/33059 PCT/US95/06530 (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.

412, 1982). A particularly useful bacterial expression system employs the phage X PL promoter and c1857ts thermoinducible repressor. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 (ATCC 53082).

The recombinant receptor protein may also be expressed in yeast hosts, preferably from Saccharomyces species, such as S. cerevisiae. Yeast of other genera such as Pichia or Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2.tm yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the receptor fusion protein, sequences for polyadenylation and transcription termination and a selection gene. Preferably, yeast vectors will include an origin of replication and selectable markers permitting transformation of both yeast and E.

coli, the ampicillin resistance gene of E. coli and the S. cerevisiae trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and a promoter derived from a highly expressed yeast gene to induce transcription of a structural sequence downstream. The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and a-factor secretion leader.

The ADH2 promoter has been described by Russell et al. Biol. Chem. 258:2674, 1982) and Beier et al., (Nature 300:724, 1982). The yeast a-factor leader, which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. See, Kurjan et al., Cell 30:922, 1982; and Bitter et al., Proc.

Natl. Acad. Sci. USA 81:5330, 1984. The leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.

r _1 -I i WO 95/33059 PCT/US95/06530 Suitable yeast transformation protocols are known to those of skill in the art. An exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, (1978), selecting for Trp+ transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 pg/ml adenine and 20 gg/ml uracil.

Host strains transformed by vectors comprising the ADH2 promoter may be grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 jig/ml adenine and 80 gg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4'C prior to further purification.

Various mammalian or insect cell culture systems can be employed to express recombinant protein. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

Examples of suitable mammalian host cell lines include L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Additional suitable mammalian host cells include CV-1 cells (ATCC CCL70) and COS-7 cells (ATCC CRL 1651; described by Gluzman, Cell 23:175, 1981), both derived from monkey kidney. Another monkey kidney I cell line, CV-1/EBNA (ATCC CRL 10478), was derived by transfection of the CV-1 cell iline with a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) and with a vector containing CMV regulatory sequences (McMahan et al., EMBO 10:2821, 1991). The j 20 EBNA-1 gene allows for episomal replication of expression vectors, such as HAV-EO or pDC406, that contain the EBV origin of replication.

Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a poly-adenylation site, splice donor and acceptor sites, and transcriptional termination sequences. The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin or replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the BglI site located in the viral origin of replication is included.

13 -rc; Y WO 95/33059 PCT/US95/06530 Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol.

Cell. Biol. 3:280, 1983). One useful system for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). Vectors derived from retroviruses also may be employed.

When secretion of the OSM-RP protein from the host cell is desired, the expression vector may comprise DNA encoding a signal or leader peptide. In place of the native signal sequence, a heterologous signal sequence may be added, such as the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.

The present invention provides a process for preparing the recombinant proteins of the present invention, comprising culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes said protein under conditions that promote expression. The desired protein is then purified from culture media or cell extracts. The desired protein may be OSM-RB or the heterodimeric receptor, for example. Cell-free translation systems could also be employed to produce the desired protein using RNA derived from the novel DNA of the present invention.

As one example, supernatants from expression systems that secrete recombinant protein into the culture medium can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix can comprise oncostatin M. An oncostatin M affinity matrix may be prepared by coupling recombinant human oncostatin M to cyanogen bromide-activated Sepharose (Pharmacia) or Hydrazide Affigel (Biorad), according to manufacturer's recommendations. Sequential immunopurification using antibodies bound to a suitable support is preferred. Proteins binding to an antibody specific for OSM-RB are recovered and contacted with antibody specific for gp130 on an insoluble support. Proteins immunoreactive with both antibodies may thus be identified and isolated.

SAlternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. One or more reversed-phase high performance -"LL -~i~i~lLW~Lli~l I IWL I1IL IUUU LU VnAUUU ULIIMN C1uuIVUs, CoIT]poII it::[tiw, 11 uly u ui N T C WINWORDUACKIENODELETESP26016.DOC WO 95/33059 PCT/US95/06530 liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a fusion protein.

Some or all of the foregoing purification steps, in various combinations, can be employed to provide an essentially homogeneous recombinant protein. Recombinant cell culture enables the production of the fusion protein free of those contaminating proteins which may be normally associated with gpl30 or OSM-RB as they are found in nature in their respective species of origin, on the surface of certain cell types.

The foregoing purification procedures are among those that may be employed to purify non-recombinant receptors of the present invention as well. When linking procedures that may produce homodimers (gp 130-linker-gp130 and OSM-RB-linker-OSM- RB) are employed, purification procedures that separate the heterodimer from such homodimers are employed. An example of such a procedure is sequential immunopurification as discussed above. In one embodiment, OSM-RB (recombinant or non-recombinant) is purified such that no bands corresponding to other (contaminating) proteins are detectable by SDS-PAGE.

Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant fusion proteins can disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express fusion proteins as a secreted protein greatly simplifies purification. Secreted recombinant protein resulting from a large-scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J.

Chromatog. 296:171, 1984), involving two sequential, reversed-phase HPLC steps for purification of a recombinant protein on a preparative HPLC column.

The DNA or amino acid sequences of gpl30 and OSM-RB may vary from those presented in SEQ ID NO:1 and SEQ ID NO:5, respectively. Due to the known degeneracy of the genetic code, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence. In addition, DNA sequences capable of hybridizing to the native DNA sequence of SEQ ID NO:1 or SEQ ID NO:5 under moderately stringent or highly stringent conditions, and which encode a biologically active gpl30 or OSM-RB polypeptide, respectively, are also considered to be gpl30-encoding or OSM-RB-encoding DNA sequences, in the context of the present invention. Such hybridizing sequences include but are not limited to variant sequences such as those described below, and DNA derived from other mammalian species. Human OSM-RB is within the scope of the present r.' WO 95/33059 PCT/US95/06530 invention, as are OSM-R3 proteins derived from other mammalian species, including but not limited to rat, bovine, porcine, or various non-human primates.

Moderately stingent conditions include conditions described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1, pp 1.101-104, Cold Spring Harbor Laboratory Press, 1989. Conditions of moderate stingency, as defined by Sambrook et al., include use of a prewashing solution of 5X SSC, 0.5% SDS, mM EDTA (pH 8.0) and hybridization conditions of about 55C, 5 X SSC, overnight.

Highly stringent conditions include higher temperatures of hybridization and washing. The skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the probe. One embodiment of the invention is directed to DNA sequences that will hybridize to the OSM- RB DNA of SEQ ID NO:5 under highly stringent conditions, wherein said conditions include hybridization at 68 0 C followed by washing in 0.1X SSC/0.1% SDS at 63-68 0 C. In another embodiment, the present invention provides a heterodimeric receptor comprising OSM-RB and gpl30, wherein said OSM-RB and gp130 are encoded by DNA that hybridizes to the DNA of SEQ ID NO:5 or SEQ ID NO:1, respectively, under moderately or highly stringent conditions.

Further, certain mutations in a nucleotide sequence which encodes OSM-RB or will not be expressed in the final protein product. For example, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see EP 75,444A). Other alterations of the nucleotide sequence may be made to provide codons that are more readily translated by the selected host, the well-known E. coli preference codons for E. coli expression.

The amino acid sequence of native gpl30 or OSM-RB may be varied by substituting, deleting, adding, or inserting one or more amino acids to produce a gpl30 or OSM-RB variant. Variants that possess the desired biological activity of the native and OSM-RB proteins may be employed in the receptor of the present invention. Assays by which the biological activity of variant proteins may be analyzed are described in the examples below. Biologically active gpl30 polypeptides are capable of binding oncostatin M. The desired biological activity of the OSM-RB polypeptides disclosed herein is the ability to enhance the binding of oncostatin M when OSM-RB is joined to gpl30, compared to the level of oncostatin M binding to gpl30 alone.

Alterations to the native amino acid sequence may be accomplished by any of a number of known techniques. For example, mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

16 WO 95/33059 PCT/US95/06530 Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craig (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); U.S. Patent No. 4,518,584, and U.S. Patent No. 4,737,462, which are incorporated by reference herein.

Bioequivalent variants of OSM-RB and gp 130 may be constructed by, for example, making various substitutions of amino acid residues or deleting terminal or internal amino acids not needed for biological activity. In one embodiment of the invention, the variant amino acid sequence is at least 80% identical, preferably at least 90% identical, to the native sequence. Percent similarity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482, 1981). Briefly, the GAP program defines similarity as the number of aligned symbols nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and no penalty for end gaps.

Generally, substitutions should be made conservatively; the most preferred substitute amino acids are those having physiochemical characteristics resembling those of the residue to be replaced. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.

Cysteine residues can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.

Hydrophilic amino acids may be substituted for hydrophobic amino acids in the transmembrane region and/or intracellular domain of gp130 and OSM-RB to enhance water solubility of the proteins.

WO 95/33059 PCT/US95/06530 Adjacent dibasic amino acid residues may be modified to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. These amino acid pairs, which constitute KEX2 proteases processing sites, are found at residues 290-291, 291-292, 580-581, and 797-798 of the OSM-RB protein of SEQ ID NO:6. These KEX2 sites are found at positions 153-154 and 621-622 of the protein of SEQ ID NO:2. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites.

The present invention also includes proteins with or without associated nativepattern glycosylation. Expression of DNAs encoding the fusion proteins in bacteria such as E. coli provides non-glycosylated molecules. Functional mutant analogs having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems.

N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn- A1-Z, where Al is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate.

The OSM-RB amino acid sequence in SEQ ID NO:6 contains 16 such Nglycosylation sites, all found in the extracellular domain, at amino acids 15-17, 57-59, 104- 106, 136-138, 149-151, 194-196, 280-282, 299-301, 318-320, 334-336, 353-355, 395- 397, 419-421, 464-466, 482-484, and 553-555 of SEQ ID NO:6. The extracellular domain of gpl30 comprises N-glycosylation sites at positions 21-23, 61-63, 109-111, 135-137, 205-207, 224-226, 357-359, 361-363, 368-370, 531-533, and 542-544 of SEQ ID NO:2. Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between Al and Z, or an amino acid other than Asn between Asn and Al. Known procedures for inactivating Nglycosylation sites in proteins include those described in U.S. Patent 5,071,972 and EP 276,846.

Variants of the receptor proteins of the present invention also include various structural forms of the primary protein which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, a receptor protein may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction.

The primary amino acid structure also may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, 18 WO 95/33059 PCT/US95/06530 phosphate, acetyl groups and the like. Covalent derivatives are prepared by linking particular functional groups to amino acid side chains or at the N- or C- termini. Other derivatives of the receptor protein within the scope of this invention include covalent or aggregative conjugates of the receptor protein with other proteins or polypeptides, such as by synthesis in recombinant culture as N- or C- terminal fusions. For example, the conjugated polypeptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall the yeast a-factor leader).

Peptides may be fused to the desired protein via recombinant DNA techniques) to facilitate purification or identification. Examples include poly-His or the Flag® peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:7) (Hopp et al., BiolTechnology 6:1204, 1988, and U.S. Patent 5,011,912). The Flag® peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. Expression systems useful for fusing the Flag® octapeptide to the N- or C-terminus of a given protein are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, CT, as are monoclonal antibodies that bind the octapeptide.

Encompassed by the present invention are OSM-RB polypeptides in the form of oligomers, such as dimers or trimers. Such oligomers may be naturally occurring or produced by recombinant DNA technology. The present invention provides oligomers of OSM-RB (preferably the extracellular domain or a fragment thereof), linked by disulfide bonds or expressed as fusion proteins with or without peptide linkers. Oligomers may be formed by disulfide bonds between cysteine residues on different OSM-RB polypeptides, for example. In another embodiment, OSM-RB oligomers may be prepared using polypeptides derived from immunoglobulins, as described above.

Naturally occurring OSM-RB variants are also encompassed by the present invention. Examples of such variants are proteins that result from alternative mRNA splicing events or from proteolytic cleavage of the OSM-RB protein, wherein the desired biological activity is retained. Alternative splicing of mRNA may yield a truncated but biologically active OSM-RB protein, such as a naturally occurring soluble form of the t *protein, for example. Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the OSM-RB protein (generally from 1-5 terminal amino acids). Naturally occurring gpl30 variants may be employed in the inventive receptors.

The present invention also provides a pharmaceutical composition comprising a receptor protein of the present invention with a physiologically acceptable carrier or dilient.

I

I- WO 95/33059 PCT/US95/06530 Such carriers and diluents will be nontoxic to recipients at the dosages and concentrations employed. Such compositions may, for example, comprise the receptor protein in a buffered solution, to which may be added antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. The receptor of the present invention may be administered by any suitable method in a manner appropriate to the indication, such as intravenous injection, local administration, continuous infusion, sustained release from implants, etc.

The heterodimeric receptor of the present invention (comprising gpl30 and OSM- RB) is useful as an oncostatin M binding reagent. This receptor, which preferably comprises soluble gpl30 and soluble OSM-RB, has applications both in vitro and in vivo.

The receptors may be employed in in vitro assays, in studies of the mechanism of transduction of the biological signal that is initiated by binding of oncostatin M to this receptor on a cell. Such receptors also could be used to inhibit a biological activity of oncostatin M in various in vitro assays or in vivo procedures. In one embodiment of the invention, the inventive receptor is administered to bind oncostatin M, thus inhibiting binding of the oncostatin M to endogenous cell surface receptors. Biological activity mediated by such binding of oncostatin M to the cells thus is also inhibited.

gpl30 alone binds oncostatin M, but with relatively low affinity (Gearing et al., Science 255:1434, 1992). Heterodimeric receptors comprising a leukemia inhibitory factor (LIF) receptor and gpl30 bind oncostatin M with higher affinity than does gpl30 alone, but also bind LIF with high affinity (Gearing et al., supra). Receptors of the present invention, produced by cells co-transfected with OSM-RB- and gpl30-encoding DNA, for example, bind oncostatin M with high affinity but do not function as a high affinity LIF receptors. Such receptors of the present invention may be employed when inhibition of an oncostatin M-mediated activity, but not a LIF-mediated activity, is desired, for example.

Oncostatin M shares certain properties with LIF, but exhibits other activities that are not exhibited by LIF. In addition, use of the receptors of the present invention in vitro assays offers the advantage of allowing one to determine that the assay results are attributable to binding of oncostain M, but not LIF, by the receptor.

In one embodiment of the invention, a heterodimeric receptor comprising OSM-RB and gpl30 is administered in vivo to inhibit a biological activity of oncostatin M.

Oncostatin M has exhibited growth modulating activity on a variety of different cell types, and has been reported to stimulate hematopoiesis, stimulate epithelial cell proliferation, increase plasmin activity (thereby inducing fibrinolysis), inhibit angiogenesis and supress expression of major histocompatibility complex antigens on endothelial cells. See PCT application WO 9109057 and European patent application no. 422,186. When these or ii, IIPILP 3 17 WO 95/33059 PCT/US95/06530 other biological effects of oncostatin M are undesirable, a receptor of the present invention may be administered to bind oncostatin M.

The inventive receptor may be administered to a patient in a therapeutically effective amount to treat a disorder mediated by oncostatin M. A disorder is said to be mediated by oncostatin M when oncostatin M causes (directly or indirectly) or exacerbates the disorder.

Soluble receptor proteins can be used to competitively bind to oncostatin M, thereby inhibiting binding of oncostatin M to endogenous cell surface receptors. Oncostatin M is believed to stimulate production of the cytokine interleukin-6 as reported by Brown et al., J. Immunol. 147:2175 (1991). Oncostatin M therefore may indirectly mediate disorders associated with the presence of IL-6. IL-6 has been reported to be involved in the pathogenesis of AIDS-associated Kaposi's sarcoma (deWit et al., J. Intern. Med.

[England] 229:539, 1991). Oncostatin M has been reported to play a role in stimulating I proliferation of Kaposi's sarcoma cells (Nair et al., Science 255:1430, 1992, and Miles et al., Science 255:1432, 1992). Binding of oncostatin M by a receptor of the present invention (preferably a soluble form thereof) thus may be useful in treating Kaposi's sarcoma.

Heterodimeric receptors comprising OSM-RB linked to gp130 also find use in assays for biological activity of oncostatin M proteins, which biological activity is measured in terms of binding affinity for the receptor. To illustrate, the receptor may be employed in a binding assay to measure the biological activity of an oncostatin M fragment, variant, or mutein. The receptor is useful for determining whether biological activity of oncostatin M is retained after modification of an oncostatin M protein chemical modification, mutation, etc.). The binding affinity of the modified oncostatin M protein for the receptor is compared to that of an unmodified oncostatin M protein to detect any adverse impact of the modification on biological activity. Biological activity thus can be assessed before the modified protein is used in a research study or assay, for example.

The heterodimeric receptors also find use as reagents that may be employed by those conducting "quality assurance" studies, to monitor shelf life and stability of oncostatin M proteins under different conditions. The receptors may be used to confirm biological activity (in terms of binding affinity for the receptor) in oncostatin M proteins that have been stored at different temperatures, for different periods of time, or which have been produced in different types of recombinant expression systems, for example.

The present invention further provides fragments of the OSM-Rp nucleotide sequences presented herein. Such fragments desirably comprise at least about 14 nucleotides of the sequence presented in SEQ ID NO:5. DNA and RNA complements of said fragments are provided herein, along with both single-stranded and double-stranded forms of the OSM-Rp DNA.

WO 95/33059 PCT/US95/06530 Among the uses of such nucleic acid fragments is use as a probe. Such probes may be employed in cross-species hybridization procedures to isolate OSM-Rp DNA from additional mammalian species. As one example, a probe corresponding to the extracellular domain of OSM-Rp may be employed. The probes also find use in detecting the presence of OSM-Rp nucleic acids in in vitro assays and in such procedures as Northern and Southern blots. Cell types expressing OSM-Rp can be identified. Such procedures are well known, and the skilled artisan can choose a probe of suitable length, depending on the particular intended application. The probes may be labeled with 32 P) by conventional techniques.

Other useful fragments of the OSM-Rp nucleic acids are antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target OSM-Rp mRNA (sense) or OSM-Rp DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, may comprise a fragment of the coding region of OSM-Rp cDNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides.

The ability to create an antisense or a sense oligonucleotide based upon a cDNA sequence for a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block translation (RNA) or transcription (DNA) by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of OSM-Rp proteins.

Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as Sthose described in WO 90/10448, and other moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the.

antisense or sense oligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO 4 mediated DNA transfection, electroporation, or by using gene transfer vectors such as -Ji I I m m WO 95/33059 PCT/US95/06530 Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retroviral vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated and DCT5C (see PCT Application US 90/02656).

Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.

Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.

The following examples are provided to illustrate certain embodiments of the invention, and are not to be construed as limiting the scope of the invention.

EXAMPLES

Example 1 Isolation of DNA Encoding OSM-RB DNA encoding the B subunit of the oncostatin M receptor was isolated as follows.

The procedure began with preparation of oligonucleotides degenerate to amino acid sequences that are conserved among proteins of the hematopoietin receptor family.

Alignment of the amino acid sequences of three proteins in the hematopoietin receptor family (gpl30, LIF receptor, and G-CSF receptor) reveals several highly conserved regions. Such conserved regions are identified and discussed by Gearing et al.

in Polyfunctional Cytokines: IL-6 and LIF, Bock et al., Eds., John Wiley Sons, Chichester, UK, 1992, page 245. After including homologous sequences from the ychain of the IL-2 receptor as well (Takeshita et al. Science 257:379, 1992), oligonucleotides degenerate to certain of the conserved regions sets of oligonucleotides that include all possible DNA sequences that can encode the amino acid sequences in the conserved regions) were prepared by conventional techniques.

Two sets of degenerate oligonucleotides were used as primers in a polymerase chain reaction (PCR). 5' primers were degenerate to the amino acid sequence P K- WO 95/33059 PCT/US95/06530 PheArgXArgCys (SEQ ID NO:9), which is found at positions 275-279 of the gp130 sequence of SEQ ID NO:2, wherein X represents lie (found at that position in gpl30 and LIF-R) or Val (for IL-2Ry). Additional 5' primers degenerate to the sequence LeuGlnIleArgCys (SEQ ID NO:10), which is found at the corresponding position in G- CSF-R, were employed as well. The 3' primers were degenerate to the amino acid sequence TrpSerXTrpSer (SEQ ID NO:11), which is found at positions 288-292 of the sequence of SEQ ID NO:2, wherein X represents Asp (found at that position in and G-CSF-R), Lys (for LIF-R), or Glu (for IL-2Ry).

To test the viability of this approach, PCR was conducted using the abovedescribed primers with LIF-R, gpl30, G-CSF-R, or IL-2RY DNA as the template. The reactions were conducted by conventional techniques, and the reaction products were analyzed by gel electrophoresis. For each reaction, a band about 50 base-pairs in size was seen on the gel, indicating successful amplification of a DNA fragment of the expected size.

PCR was then conducted using genomic human DNA as the template. The reaction products were analyzed by gel electrophoresis, and a 50bp band was visualized. This band was excised from the gel, and the DNA was eluted therefrom. The DNA was subcloned into the cloning vector pBLUESCRIPT® SK, which is available from Stratagene Cloning Systems, La Jolla, California. E. coli cells were transformed with the resulting recombinant vectors, and individual colonies of the transformants were cultivated in 96well plates.

Twelve colonies were chosen at random, and the recombinant vectors were isolated therefrom. The nucleotide sequences of the DNA inserts of the vectors were determined.

Seven of these inserts were identified by their sequence as gp130 DNA, two were LIF-R, one contained a stop codon and did not appear to be of interest, and two contained a novel sequence (the same sequence, in both orientations). An oligonucleotide probe containing this novel sequence (the portion of the insert that is between the two primer sequences) was prepared and labeled with 32 P by standard techniques.

The 32 P-labeled probe was used to screen two different cDNA libraries, one derived from human placenta and the other from a cell line designated IMTLH-1. The placental library was chosen because placenta is a rich source of growth and differentiation factors. The IMTLH cells, obtained by transformation of human bone marrow stromal cells with pSV-neo, were chosen because they were found to bind oncostatin M but not LIF (Thoma et al., J. Biol. Chem. 269:6215, 1994). In addition, an RNA band of about 5.5-6.0 kb was detected on Northern blots of RNA derived from IMTLH-1 cells and placenta, probed with the above-identified 32 p-labeled probe.

Positive clones were isolated from both libraries and determined by DNA sequencing to contain various portions of the novel DNA of interest. Although an initiator v, ,u rn JO/lo) ana tac promoter 11 WO 95/33059 PCT/US95/06530 codon (indicating the 5' end of a coding region) was identified, none of the clones appeared to contain the stop codon that would represent the 3' end of the coding region.

An oligonucleotide probe corresponding to sequence found near the 3' end of several of the clones was synthesized and labeled with 32 P by standard techniques. The probe was used to screen a cDNA library derived from the SV40-transformed human lung fibroblast cell line WI-26 VA4. This library was constructed as described in example 2 of U.S. Patent 5,264,416, which is hereby incorporated by reference. Clones comprising additional coding sequence at the 3' end (compared to the previously-identified clones above) were isolated.

An expression vector was constructed, containing a DNA fragment comprising this 3' end of the novel sequence ligated to DNA fragments from the above-described clones containing the 5' end of the novel sequence. The nucleotide sequence of the human OSM- RB DNA in the resulting recombinant vector is presented in SEQ ID NO:5. The protein encoded by the isolated DNA is presented in SEQ ID NO:6.

The vector was a mammalian expression vector designated pDC409. This vector is similar to pDC406, described in McMahan et al., (EMBO J. 10:2821, 1991). A Bgl II site 4 outside the multiple cloning site (mcs) in pDC406 has been deleted so that the BglII site in i the mcs of pDC409 is unique. The pDC409 multiple cloning site (mcs) differs from that of pDC406 in that it contains additional restriction sites and three stop codons (one in each reading frame). A T7 polymerase promoter downstream of the mcs facilitates sequencing of DNA inserted into the mcs.

The OSM-RB cDNA insert was excised from an expression vector using restriction enzymes that cleave within the 5' and 3' non-coding regions of the cDNA. The excised cDNA was ligated into the EcoRV site of the cloning vector pBluescript® SK- (Stratagene Cloning Systems, LaJolla, CA). The Eco RV site, found in the multiple cloning site of the vector, was destroyed by insertion of the cDNA. E. coli cells transformed with the resulting recombinant vector were deposited with the American Type Culture Collection, Rockville, MD, on August 16, 1994, and assigned accession no. ATCC 69675.

The deposit was made under the terms of the Budapest Treaty.

The encoded OSM-RB amino acid sequence presented in SEQ ID NO:6 comprises San N-terminal signal peptide (amino acids -27 to followed by an extracellular domain S(amino acids 1 to 714), a transmembrane region (amino acids 715 to 734) and a cytoplasmic domain (amino acids 735 to 952). The OSM-RB amino acid sequence is approximately 30% identical to that of the LIF receptor protein described in Gearing et al.

(EMBO J. 10:2839, 1991) and in U.S. Patent 5,284,755, hereby incorporated by reference. The DNA sequence of the coding region of OSM-RB is about 48% identical to the portion of LIF-R DNA that aligns with the OSM-RB coding region when the abovedescribed GAP computer program is employed.

cc WO 95/33059 PCT/US95/06530 Example 2 Assay to Detect Binding of Oncostatin M An assay for binding of oncostatin M by cells expressing both recombinant and recombinant OSM-RB was conducted as follows. An assay for oncostatin M binding by cells expressing gpl30 alone was also conducted for purposes of comparison.

Oncostatin M may be purified from cells in which the protein is naturally found, or from cells transformed with an expression vector encoding oncostatin M. One source of oncostatin M is phorbol ester-treated U937 cells, as described by Zarling et al., PNAS U.SA. 83:9739 (1986). Purification of recombinant oncostatin M is described by Linsley et al. Biol. Chem. 264:4282-4289, 1989) and Gearing et al. (EMBO J. 10:2839, 1991).

Oncostatin M (OSM) may be radiolabeled using any suitable conventional procedure. Radioiodination of oncostatin M has been described by Linsley et al., supra., for example. In one suitable procedure, OSM is radiolabeled using a commercially available enzymobead radioiodination reagent (BioRad) according to manufacturer's instructions. The resulting 125 1-OSM is diluted to a working stock solution in binding medium, which is RPMI 1640 medium containing 2.5% bovine serum albumin (BSA), 0.2% sodium azide, and 20 mM Hepes, pH 7.4.

CV1-EBNA-1 cells in 150 mm dishes (3.6 x10 6 cells/dish) were transfected with a expression vector, or were co-transfected with the gpl30-encoding vector and an OSM-RB-encoding vector. All cells were additionally co-transfected with a mammalian expression vector designated pDC410, described below.

The OSM-RB-encoding vector was the recombinant vector described in example 1, comprising full length OSM-RB DNA in mammalian expression vector pDC409. The vector comprised the human gpl30 DNA sequence of SEQ ID NO:1 in a mammalian expression vector designated pDC304. A similar recombinant vector, comprising the same gpl30-encoding DNA in mammalian expression vector pDC303, was deposited in E. coli strain DH5a host cells with the American Type Culture Collection, Rockville, Maryland. These transformed cells were deposited under the name B10G/pDC303 (DH5a) on November 14, 1991 and assigned ATCC Accession No.

i 68827. The deposit was made under the terms of the Budapest Treaty.

pDC304 comprises a NotI site in its multiple cloning site, but is otherwise identical to pDC303. pDC304 also is essentially identical to pCAV/NOT, described in PCT application WO 90/05183, except that a segment of the adenovirus-2 tripartite leader (TPL) containing a cryptic promoter functional in bacteria has been deleted. Protein expression from the cryptic promoter is potentially disadvantageous for preparing and isolating a desired recombinant plasmid in bacterial cells.

I_ -~tY1;-"araxrP~~lre;ar;~:~~r SWO 95/33059 PCT/US95/06530 The pDC410 vector is identical to the pDC409 vector described in example 1, except that the EBV origin of replication of pDC409 is replaced by DNA encoding the large T antigen driven from the SV40 promoter in pDC410. Co-transfecting the cells with this vector provides the SV40 T-antigen that drives high level DNA replication of the other plasmid vectors, which contain the SV40 origin of replication. pDC410 thus is important for episomal replication of the co-transfected vectors in CV1-EBNA-1 cells.

The transfected cells were cultured for 24 hours, trypsinized and replated, then cultured another 24 hours to permit expression of the encoded proteins, which were retained on the cell membrane. The adherent cells were dislodged using 5mM EDTA in PBS, then washed twice with binding medium (RPMI 1640 medium containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, and 20 mM HEPES, pH The cells then were incubated with various concentrations of 125 I-labeled oncostatin M in binding medium for 1 hour at 370C with gentle agitation.

Free and cell-bound 125 I-oncostatin M were separated using the phthalate oil separation method of Dower et al. (J Immunol. 132:751, 1984), essentially as described by Park et al. Biol. Chem. 261:4177, 1986, and Proc. Natl. Acad. Sci. USA 84:5267, 1987). The free and cell-bound 125 I-oncostatin M were quantified on a Packard Autogamma Counter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) were generated on RS/1 (BBN Software, Boston, MA) run on a Microvax computer.

The results are presented in Figures 1 and 2, in the form of Scatchard analyses.

Figure 1 presents the results for cells expressing gpl30 alone. These transfected cells exhibited a single affinity class of binding, with approximately 29,310 receptor sites per cell, and an affinity constant (Ka) of 2.64 x 108. Figure 2 presents the results for cells expressing gpl30 and OSM-RB. A biphasic pattern can be seen, indicating two binding components. The first component (approximately 2196 receptor sites per cell) exhibited an affinity constant of 7.18 x 109. The second component (approximately 36,471 receptor sites per cell) exhibited an affinity constant of 2.34 x 108. Thus, a relatively high affinity binding component is seen in the cells expressing both gp130 and OSM-RB. These high affinity binding sites were absent in the cells expressing gpl30 alone.

The cells co-transfected with both OSM-RB- and gpl30-encoding expression vectors expressed a receptor protein of the present invention. The receptor binds oncostatin M with higher affinity than does the gp130 protein expressed on cells transfected with the vector alone.

27 WO 95/33059 PCT/US95/06530 Example 3 Preparation of Monoclonal Antibodies Directed Against OSM-RB Purified OSM-RB polypeptides of the present invention are employed as immunogens to generate monoclonal antibodies immunoreactive therewith using conventional techniques, for example, those disclosed in U.S. Patent 4,411,993. Suitable immunogens include, but are not limited to, full length recombinant OSM-RB or fragments thereof, such as the extracellular domain. To immunize mice, the immunogen is emulsified in complete Freund's adjuvant and injected subcutaneously in amounts ranging from 100 pg into Baib/c mice. Ten to twelve days later, the immunized animals are boosted with additional immunogen emulsified in incomplete Freund's adjuvant and periodically boosted thereafter on a weekly to biweekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay (antibody sandwich) or ELISA (enzyme-linked immunosorbent assay). Other assay procedures are also suitable.

Following detection of an appropriate antibody titer, positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to a murine myeloma cell line, NS 1 or, preferably, P3x63Ag8.653 (ATCC CRL 1580). Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a HAT selective medium (hypoxantine, aminopterin, and thymidine) to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivity with the receptor protein, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem 8.871 (1971) and in U.S. Patent 4,704,004. A preferred screening technique is the antibody capture technique described in Beckmann et al. Immunol.

144:4212, 1990). Positive clones are then injected into the peritoneal cavities of syngeneic Balb/c mice to produce ascites containing high concentrations (greater than 1 mg/ml) of anti-OSM-RB monoclonal antibody. The resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography, and/or affinity chromatography based on binding of antibody to Protein A of Staphylococcus aureus.

Example 4 Receptors Comprising gpl30 Polypeptides Lacking FNIII Domains DNA sequences encoding soluble gpl30 proteins lacking fibronectin type III (FNIII) domains were isolated and fused to an Fc-encoding sequence. Deleting the FNIII domains affords the advantage of reducing the size of the gpl30/Fc fusion protein, WO 95/33059 PCT/US95/06530 contains three FNIII domains, comprising amino acids 300 (Tyr) to 399 (Phe), 400 (Gin) to 496 (Pro), and 497 (Pro) to 597 (Glu), respectively, of SEQ ID NO:2. From one to all three of the FNIII domains may be removed from gp 130 to teduce the size of the protein.

The FNIII domains of gpl30 were removed by digesting a recombinant encoding expression vector with BstX1, then blunting the overhang using T4 DNA polymerase according to conventional procedures. The recognition site for BstX1 spans nucleotides 1231-1242 of SEQ ID NO:1 (gp130), cleaving within the codons for amino acids 10-11 of the first FNIII domain of gpl30. The cleaved vector was then digested with which cleaves within the polylinker of the vector upstream of the Fc sequence and generates blunt ends. The (BstX1)/EcoR5 fragment comprising a sequence encoding the end of gpl30 (lacking the FNIII domains), the vector sequences, the Fc sequence, and a portion of the polylinker, was ligated to recircularize the vector.

E. coli cells were transformed with the ligation mixture, plasmids were isolated therefrom, and the desired recombinant plasmid was identified by restriction analysis. The fusion protein encoded by the construct comprises (from N- to C-terminus) amino acids -22 to 308 of SEQ ID NO:2 (gpl30), a four amino acid spacer peptide -Asn-Arg-Tyr-Valencoded by the polylinker segment, and amino acids 1-232 of SEQ ID NO:3 The polypeptide moiety contains the first 9 amino acids of the first FNIII domain, but lacks the remainder of the first FNIII domain and all of the second and third FNIII domains.

A heterodimeric receptor of the present invention may comprise OSM-RB and the foregoing truncated gpl30 polypeptide lacking FNIII domains. COS-7 cells or other suitable host cells are co-transfected with OSM-RB-encoding and truncated expression vectors. The co-transfected cells are cultured to express the heterodimeric receptor.

EXAMPLE Assay for Binding of Oncostatin M and LIF by Receptors An assay for binding of oncostatin M or leukemia inhibitory factor (LIF) by various receptor proteins was conducted as follows. The receptor proteins included soluble OSM- Rp/Fc, gpl30/Fc, LIF-R/Fc, and combinations thereof. Results of the assay are presented in Figure 3.

An expression vector encoding a soluble OSM-Rp/Fc fusion protein, which comprised a truncated extracellular domain of OSM-Rp fused to the N-terminus of an Fc region polypeptide derived from an antibody, was constructed as follows. The recombinant expression vector prepared in example 1, comprising OSM-Rp DNA in vector pDC409, was digested with the restriction enzyme SphI, treated with T4 DNA polymerase WO 95/33059 PCTIUS95/06530 to remove the 3' overhangs (generating blunt ends), then digested with Sal I, which cleaves upstream of the OSM-RB coding region. The desired fragment, which includes the 5' end of the OSM-RB DNA, terminating at nucleotide 1744 of SEQ ID NO:5, was isolated by conventional techniques.

A recombinant vector designated hIgG lFc comprises the Fc polypeptide-encoding cDNA of SEQ ID NO:3, as described above. Vector hIgGlFc was digested with the restriction enzymes Sna B1 and NotI, which cleave in the polylinker region of the vector, upstream and downstream, respectively, of the Fc polypeptide-encoding cDNA.

The thus-isolated Fc polypeptide-encoding DNA fragment and the OSM-Rpencoding DNA fragment isolated above were ligated into a SalI/NotI-digested expression vector pDC304 such that the Fc polypeptide DNA was fused to the 3' end of the OSM-Rp DNA. The mammalian expression vector pDC304 is described in example 2. The resulting expression vector encoded a fusion protein comprising amino acids -27 through 432 of the OSM-Rp sequence of SEQ ID NO:6, followed by a valine residue encoded by a vector polylinker segment, followed by amino acids 1 through 232 of the Fc polypeptide sequence of SEQ ID NO:4.

An expression vector encoding a soluble human gpl30/Fc fusion protein was constructed as follows. Recombinant vector B10G/pDC303 (ATCC 68827) comprising human gpl30 cDNA was digested with EcoR1, and the resulting 5' overhang was rendered blunt using T4 DNA polymerase. The recognition site for EcoR1 comprises nucleotides 2056-2061 of SEQ ID NO:1. The EcoRl-digested vector was then cleaved with XhoI, which cleaves in the vector upstream of the gpl30 cDNA insert.

Vector hIgG1Fc, comprising Fc polypeptide-encoding cDNA as described above, was digested with StuI (a blunt cutter) and NotI, which cleave upstream and downstream, respectively, of the inserted Fc cDNA. The XhoI/(EcoR1) gpl30 fragment isolated above was ligated to the Fc-containing fragment and to XhoI/NotI-digested mammalian expression vector pDC304.

E. coli cells were transformed with the ligation mixture, plasmids were isolated therefrom by conventional procedures, and the desired recombinant vector was identified by restriction analysis. The gpl30/Fc fusion protein encoded by the recombinant vector comprises (from N- to C-terminus) amino acids -22 to 582 of SEQ ID NO:2 Sfollowed by 7 amino acids constituting a peptide linker encoded by the polylinker segment of plasmid hIgGlFc, followed by amino acids 1-232 of SEQ ID NO:4 (Fc).

An expression vector encoding a soluble human LIF-R/Fc fusion protein was constructed as described in example 5 of U.S. Patent 5,284,755, hereby incorporated by reference. Briefly, a recombinant vector designated pHLIF-R-65 contains human LIF-R cDNA (a partial clone encoding a complete signal peptide, extracellular domain, and transmembrane region, and a partial cytoplasmic domain) in vector pDC303. The 1. A WO 95/33059 PCT/US95/06530 mammalian expression vector pDC303 is described in PCT application WO 93/19777. E.

coli cells transformed with pHLIF-R-65 were deposited with the American Type Culture Collection, Rockville, MD, on December 11, 1990, and assigned accession no. 68491.

DNA encoding the LIF-R signal peptide and extracellular domain (truncated at the Cterminus) was isolated and fused to DNA encoding an antibody Fc region polypeptide in pBluescript®SK The gene fusion was excised from the cloning vector and inserted into the above-described mammalian expression vector pDC304. The resulting recombinant expression vector encoded a LIF-R/Fc fusion protein comprising amino acids -44 through 702 of the LIF-R sequence presented in U.S. Patent 5,284,755, followed by a linker comprising six amino acids encoded by a vector polylinker segment, followed by amino acids 1 through 232 of the Fc amino acid sequence of SEQ ID NO:4.

CV-1-EBNA cells were transfected with one of the three recombinant expression vectors prepared above, or co-transfected with two of the vectors, as follows: Experiment Cells transfected with vector(s) encoding: A empty expression vector (control) B C LIF-R/Fc D OSM-Rp/Fc E OSM-Rp/Fc and LIF-R/Fc F OSM-Rp/Fc and G gpl30/Fc and LIF-R/Fc The transfected cells were cultured to allow expression and secretion of the fusion proteins into the culture medium. Cross-linked agarose beads bearing Protein A (Protein A Sepharose CL-4B, Pharmacia Biotech, Inc., Piscataway, NJ) were added to the culture supernatants, whereupon the fusion proteins bound to the beads via the interaction of the Fc moiety with the Protein A. Radioiodinated oncostatin M or radioiodinated LIF was also added to the culture supematants. Preparation of 1 25 1-oncostatin M is described in example 2 above. Among the known procedures for purifying and radioiodinating LIF are those described in example 1 of U.S. Patent 5,284,755. The 125 I-LIF employed in this assay Swas recombinant human LIF labeled with 125I using the enzymobead reagent (BioRad).

The culture supernatants were incubated with the Protein A beads and 125 I-LIF or 1 2 5 1-oncostatin M for 18 hours at 4 0 C. Free and cell-bound 125 I-LIF or 125 I-oncostatin M were separated by low speed centrifugation through a single step density gradient of 3% glucose in PBS. The bead-bound radioiodinated proteins were quantified on a Packard Autogamma counter.

31 WO 95/33059 PCT/US95/06530 The results are presented in Figur, 3. The bar graph in Figure 3 represents the binding of oncostatin M or LIF to the proteins expressed by cells transfected as described above for experiments A to G. The expressed proteins are bound to the Protein A beads.

Experiment A (control) revealed no significant binding of LIF or oncostatin M to proteins expressed by cells transfected with the empty expression vector pDC304. The soluble gpl30/Fc protein bound oncostatin M, but no significant binding of LIF was demonstrated (experiment The soluble LIF-R/Fc protein bound LIF, but not oncostatin M (experiment No detectable binding of LIF or oncostatin M by the soluble OSM- Rp/Fc protein was demonstrated (experiment D).

Proteins expressed by cells co-transfected with soluble LIF-R/Fc and OSM-Rp encoding vectors did not bind detectable quantities of oncostatin M, but bound LIF (experiment Proteins expressed by cells co-transfected with soluble OSM-Rp/Fc and soluble gpl30/Fc encoding vectors bound oncostatin M, but did not bind detectable quantities of LIF (experiment The binding of oncostatin M in experiment F could be inhibited by including unlabeled (cold) oncostatin M in the assay. The proteins expressed by cells co-transfected with expression vectors encoding soluble gpl30/Fc and LIF-R/Fc (experiment G) bound both oncostatin M and LIF. The LIF binding in experiment G was inhibited by adding cold LIF to the assay.

The proteins expressed when cells are co-transfected with vectors encoding soluble OSM-Rp/Fc and soluble gpl30/Fc, in accordance with the present invention, thus bind oncostatin M but not LIF. This is advantageous when binding of oncostatin M to inhibit or study a biological activity thereof) is desired, but binding of LIF is not desired.

The proteins expressed by cells co-transfected with soluble gpl30/Fc and soluble LIF-R/Fc encoding vectors bind both oncostatin M and LIF, and thus do not offer this advantageous property. In addition, cells expressing both soluble OSM-Rp/Fc and soluble bound oncostatin M at a higher level than did cells expressing soluble gp 130/Fc alone.

BRIEF DESCRIPTION OF THE SEOUENCE LISTING SEQ ID NO:1 and SEQ ID NO:2 present the DNA sequence and encoded amino acid sequence for cloned cDNA encoding an N-terminal fragment of gp 130.

SEQ ID NO:3 and SEQ ID NO:4 present the DNA sequence and encoded amino acid sequence for cloned cDNA encoding a polypeptide that corresponds to the Fc region of an IgG1 antibody.

SEQ ID NO:5 and SEQ ID NO:6 present the DNA and encoded amino acid sequence for cloned cDNA encoding the oncostatin M receptor p subunit of the present invention.

I- I WO 95/33059 PCT/US95/06530 SEQ ID NO:7 presents the amino acid sequence of a peptide that may be employed to facilitate purification of polypeptides fused thereto.

SEQ ID NO:8 presents a spacer peptide encoded by a polylinker in an expression vector, as described in example 4.

SEQ ID NOS:9, 10, and 11 are peptides that correspond to conserved sequences, as described in example 1.

WO 95/33059 PCT/US95/06530 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Mosley, Bruce Cosman, David J.

(ii) TITLE OF INVENTION: Receptor for Oncostatin M (iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Immunex Corporation STREET: 51 University Street CITY: Seattle STATE: WA COUNTRY: USA ZIP: 98101 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US 08/308,881 FILING DATE: 09-SEP-1994

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/249,553 FILING DATE: 26-MAY-1994 (viii) ATTORNEY/AGENT INFORMATION: NAME: Anderson, Kathryn A.

REGISTRATION NUMBER: 32,172 REFERENCE/DOCKET NUMBER: 2614-A (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (206) 587-0430 TELEFAX: (206) 233-0644 TELEX: 756822 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2369 base pairs S(B) TYPE: nucleic acid i STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal WO 95/33059 PCT/US95/06530 (vi) ORIGINAL SOURCE: TISSUE TYPE: human placenta (vii) IMMEDIATE SOURCE: CLONE: B10G/pDC303 (ix) FEATURE: NAME/KEY: CDS LOCATION: 244..2369 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 310..2369 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 244..309 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGCCCGCGGA GTCGCGCTGG GCCGCCCCGG CGCAGCTGAA GCCGACGGGT CTGGCCCAGC CTGGCGCCAA GGGGTTCGTG TCGAGGCGGC GCGGCCTGAG TGAAACCCAA TGGAAAAAGC ACTTAGCTTC AAATCCCTAC TCCTTCACTT ACTAATTTTG CCGGGGGCCG CGCCTGCCAG CGCTGTGGAG ACGCGGAGGG ATGACATTTA GAAGTAGAAG TGATTTGGAA ATATCCGCGC GCC TTG TTT ATT TTC Ala Leu Phe Ile Phe CCA TGT GGT TAT ATC Pro Cys Gly Tyr Ile j AAG ATG TTG ACG TTG CAG ACT TGG Met Leu Thr Leu Gin Thr Trp -22 -20 CTC ACC ACT GAA TCT ACA GGT GAA Leu Thr Thr Glu Ser Thr Gly Glu 1 CTA GTG CAA Leu Val Gin -15 CTT CTA GAT Leu Leu Asp AGT CCT GAA TCT CCA GTT Ser Pro Glu Ser Pro Val 15 TGT GTG CTA AAG GAA AAA Cys Val Leu Lys Glu Lys GTA CAA CTT CAT Val Gin Leu His AAT TTC ACT Asn Phe Thr TGT ATG GAT Cys Met Asp TTT CAT GTA AAT Phe His Val Asn GCA GTT Ala Val GCT AAT Ala Asn CAA TAT Gin Tyr TAC ATT GTC Tyr Ile Val ACT ATC ATA Thr Ile Ile ACA AAC CAT Thr Asn His ATT CCT AAG Ile Pro Lys

GAG

Glu AGA ACA GCA Arg Thr Ala GTC ACC TTT Val Thr Phe GAT ATA GCT Asp Ile Ala GGA CAG CTT Gly Gin Leu TCA TTA Ser Leu AAT ATT CAG CTC Asn Ile Gin Leu TGC AAC ATT CTT Cys Asn Ile Leu ACA TTC Thr Phe

GAA

Glu CAG AAT GTT TAT Gin Asn Val Tyr ATC ACA ATA ATT Ile Thr Ile Ile GGC TTG CCT CCA Gly Leu Pro Pro

GAA

Glu 105 WO 95/33059 PCT/US95/06530 AAA CCT AAA AAT TTG AGT TGC ATT GTG AAC GAG GGG AAG Lys Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys 110 115 TGT GAG TGG GAT GGT GGA AGG GAA ACA CAC TTG GAG ACA Cys Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr 125 130 TTA AAA TCT GAA TGG GCA ACA CAC AAG TTT GCT GAT TGC Leu Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp Cys 140 145 150 CGT GAC ACC CCC ACC TCA TGC ACT GTT GAT TAT TCT ACT Arg Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr 155 160 165 GTC AAC ATT GAA GTC TGG GTA GAA GCA GAG AAT GCC CTT Val Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu 170 175 180

AAA

Lys ATG AGG Met Arg 120 AAC TTC ACT Asn Phe Thr 135 AAA GCA AAA Lys Ala Lys GTG TAT TTT Val Tyr Phe GGG AAG Gly Lys ACA TCA GAT CAT Thr Ser Asp His

ATC

Ile 190 AAT TTT GAT CCT Asn Phe Asp Pro

GTA

Val 195 TAT AAA GTG AAG Tyr Lys Val Lys CCC AAT Pro Asn 200 CCG CCA CAT Pro Pro His TTA AAA TTG Leu Lys Leu 220

AAT

Asn 205 TTA TCA GTG ATC AAC TCA GAG GAA CTG Leu Ser Val Ile Asn Ser Glu Glu Leu TCT AGT ATC Ser Ser Ile 215 ACA TGG ACC AAC Thr Trp Thr Asn CCA AGT ATT AAG AGT GTT ATA ATA CTA Pro Ser Ile Lys Ser Val Ile Ile Leu 225 230 ACC AAA GAT GCC TCA ACT TGG AGC CAG Thr Lys Asp Ala Ser Thr Trp Ser Gin 245 AAA TAT Lys Tyr 235 AAC ATT CAA TAT Asn Ile Gin Tyr CCT CCT GAA GAC Pro Pro Glu Asp

ACA

Thr 255 GCA TCC ACC CGA Ala Ser Thr Arg TCA TTC ACT GTC Ser Phe Thr Val 912 960 1008 1056 1104 1152 1200 1248 1296 1344 GAC CTT AAA CCT TTT ACA GAA TAT GTG TTT AGG ATT CGC TGT ATG AAG Asp Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys 270 275 280 GAA GAT GGT Glu Asp Gly ATC ACC TAT Ile Thr Tyr 300 GGA TAC TGG AGT Gly Tyr Trp Ser TGG AGT GAA GAA Trp Ser Glu Glu GCA AGT GGG Ala Ser Gly 295 GAA GAT AGA CCA Glu Asp Arg Pro AAA GCA CCA AGT TTC TGG TAT AAA Lys Ala Pro Ser Phe Trp Tyr Lys 310 ATA GAT Ile Asp 315 CCA TCC CAT ACT Pro Ser His Thr GGC TAC AGA ACT Gly Tyr Arg Thr CAA CTC GTG TGG Gin Leu Val Trp ACA TTG CCT CCT Thr Leu Pro Pro GAA GCC AAT GGA AAA ATC TTG GAT TAT Glu Ala Asn Gly Lys Ile Leu Asp Tyr 340 L 1. WO 95/33059 GTG ACT CTC Val Thr Leu GCC ACA AAA Ala Thr Lys CTA ACA GTA Leu Thr Val 380 PCT/US95/06530 ACA AGA Thr Arg 350 TGG AAA TCA CAT TTA CAA AAT TAC ACA Trp Lys Ser His Leu Gin Asn Tyr Thr 355 GTT AAT Val Asn 360 ACA GTA AAT CTC Thr Val Asn Leu AAT GAT CGC TAT Asn Asp Arg Tyr CTA GCA ACC Leu Ala Thr 375 GTT TTA ACT Val Leu Thr AGA AAT CTT GTT Arg Asn Leu Val AAA TCA GAT GCA Lys Ser Asp Ala ATC CCT Ile Pro 395 GCC TGT GAC TTT Ala Cys Asp Phe GCT ACT CAC CCT GTA ATG GAT CTT AAA Ala Thr His Pro Val Met Asp Leu Lys GCA TTC CCC AAA GAT AAC ATG CTT TGG GTG GAA TGG ACT ACT CCA Ala Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro

AGG

Arg 425 GAA TCT GTA AAG Glu Ser Val Lys

AAA

Lys 430 TAT ATA CTT GAG Tyr Ile Leu Glu TGT GTG TTA TCA Cys Val Leu Ser GAT AAA Asp Lys 440 GCA CCC TGT Ala Pro Cys ACA GAC TGG CAA Thr Asp Trp Gin GAA GAT GGT ACC Glu Asp Gly Thr GTG CAT CGC Val His Arg 455 TTG ATA ACA Leu Ile Thr ACC TAT TTA AGA GGG AAC TTA Thr Tyr Leu Arg Gly Asn Leu 460 GAG AGC AAA TGC Glu Ser Lys Cys

TAT

Tyr 470 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 2064 GTT ACT Val Thr 475 CCA GTA TAT GCT Pro Val Tyr Ala GGA CCA GGA AGC CCT GAA TCC ATA AAG Gly Pro Gly Ser Pro Glu Ser Ile Lys

GCA

Ala 490 TAC CTT AAA CAA Tyr Leu Lys Gin

GCT

Ala 495 CCA CCT TCC AAA Pro Pro Ser Lys CCT ACT GTT CGG Pro Thr Val Arg AAA AAA GTA GGG Lys Lys Val Gly AAC GAA GCT GTC Asn Glu Ala Val

TTA

Leu 515 GAG TGG GAC CAA Glu Trp Asp Gin CTT CCT Leu Pro 520 GTT GAT GTT Val Asp Val ACC ATC ATT Thr Ile Ile 540 AAT GGA TTT ATC Asn Gly Phe Ile AAT TAT ACT ATA Asn Tyr Thr Ile TTT TAT AGA Phe Tyr Arg 535 TCC CAC ACA Ser His Thr GGA AAT GAA ACT Gly Asn Glu Thr GTG AAT GTG GAT Val Asn Val Asp GAA TAT Glu Tyr 555 ACA TTG TCC TCT Thr Leu Ser Ser ACT AGT GAC ACA Thr Ser Asp Thr TAC ATG GTA CGA Tyr Met Val Arg GCA GCA TAC ACA Ala Ala Tyr Thr GAA GGT GGG AAG Glu Gly Gly Lys GGT CCA GAA TTC Gly Pro Glu Phe v VO95/33059 TTT ACT ACC CCA AAG TTT GCT CAA GGA GAA Phe Thr Thr Pro Lys Phe Ala Gin Gly Giu 590 595 CCT GTT TGC TTA GCA TTC CTA TTG ACA ACT Pro Val Cys Leu Ala Phe Leu Leu Thr Thr 605 610 TGC TTT AAT AAG CGA 'AC C7'm ATT AAA AAA Cys Phe Asn Lys Arg f Ile Lys Lys] 620 625 CCA GAT CCT TCA AAG AGT CAT ATT GCC CAG Pro Asp Pro Ser Lys Ser His Ile Aia Gin 635 640 CCA AGG CAC AAT TTT AAT TCA AAA GAT CAA Pro Arg His Asn Phe Asn Ser Lys Asp Gin 650 655 TTC ACT GAT GTA AGT GTT GTG GAA ATA GAA Phe Thr Asp Val Ser Val Val Giu Ile Giu 670 675 TTT CCA GAA GAT CTG AA Phe Pro Giu Asp Leu 685 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 708 amino acids TYPE: aino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Leu Thr Leu Gin Thr Trp Leu Vai Gin -22 -20 Thr Thr Giu Ser Thr Giy Giu Leu Leu Asp 1 Pro Giu Ser Pro Val Val Gin Leu His Ser Val Leu Lys Glu Lys Cys Met Asp Tyr Phe Ile Val Trp Lys Thr Asn His Phe Thr Ile Ile Ile Asn Arg Thr Ala Ser Ser Vai Thr Leu Asn Ile Gin Leu Thr Cys Asn Ile Leu 80

%TT

Ile

CTT

Leu

CAC

H.is5

TGG

rrp

ATG

Met 660

GCA

kia PCT/US95IO6530 rC GTG 2112 al, Vai rG TTC 2160 Bu Phe bT GTT 2208 sn Vai 2T CCT 2256 ar r Pro 3C AAT 2304 ly Asn 665 %G CCT 2352 vs Pro 2369 N~O: 2: kla L Pro C: ksn P.

Hiis V Pro L- Phe T rhr P 1 WO 95/33059 Gin Pro Giu Lys Asp 155 Asn Ser Pro Lys Tyr 235 Pro Leu Asp Thr Asp 315 Thr Thr Thr Thr Thr Ile Giu His 145 Thr Giu Asp Ile Pro 225 Thr Ser Tyr Ser Ser 305 Gly Aia Ser Leu Gly 385 PCTIUS95/06530 Giu Lys 105 Arg Cys Thr Leu Lys Arg Phe Val 170 Val Thr 185 Asn Pro Ile Leu Leu Lys Gin Ile 250 Gin Asp 265 Lys Giu Giy Ile Lys Ile Trp Lys 330 Giu Vai 345 Asn Ala Thr Leu Thr Ile Pro Aia Cys Asp Phe Gin Aia Thr His Pro Vai Met Asp Leu Lys Ala D- WO 95/33059 PCT/US95/06530 Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu 415 420 425 Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 430 435 440 Pro Cys Ile Thr Asp Trp Gin Gin Glu Asp Gly Thr Val His Arg Thr 445 450 455 Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 460 465 470 Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala 475 480 485 490 Tyr Leu Lys Gin Ala Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 495 500 505 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gin Leu Pro Val 510 515 520 Asp Val Gin Asn Gly Phe Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr 525 530 535 Ile Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr Glu 540 545 550 Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 555 560 565 570 Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 575 580 585 Thr Thr Pro Lys Phe Ala Gin Gly Glu Ile Glu Ala Ile Val Val Pro 590 595 600 Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys 605 610 615 Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro 620 625 630 Asp Pro Ser Lys Ser His ile Ala Gin Trp Ser Pro His Thr Pro Pro 635 640 645 650 Arg His Asn Phe Asn Ser Lys Asp Gin Met Tyr Ser Asp Gly Asn Phe 655 660 665 Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 670 675 680 Pro Glu Asp Leu 685 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 705 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 95/33059 PCT/US95/06530 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: hIgGlFc (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..699 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAG CCC Glu Pro 1 AGA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC Arg Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys CCA GCA Pro Ala CCT GAA CTC Pro Glu Leu AAG GAC ACC Lys Asp Thr GGG GGA CCG TCA Gly Gly Pro Ser TTC CTC TTC CCC Phe Leu Phe Pro CCA AAA CCC Pro Lys Pro TGC GTG GTG Cys Val Val CTC ATG ATC TCC Leu Met Ile Ser ACC CCT GAG GTC Thr Pro Glu Val GTG GAC Val Asp GTG AGC CAC GAA GAC CCT GAG GTC AAG Val Ser His Glu Asp Pro Glu Val Lys AAC TGG TAC GTG Asn Trp Tyr Val GGC GTG GAG GTG Gly Val Glu Val AAT GCC AAG ACA Asn Ala Lys Thr CCG CGG GAG GAG Pro Arg Glu Glu TAC AAC AGC ACG Tyr Asn Ser Thr CGG GTG GTC AGC Arg Val Val Ser CTC ACC GTC CTG Leu Thr Val Leu CAC CAG His Gin GAC TGG CTG Asp Trp Leu CTC CCA GCC Leu Pro Ala 115 GGC AAG GAC TAC Gly Lys Asp Tyr TGC AAG GTC TCC Cys Lys Val Ser AAC AAA GCC Asn Lys Ala 110 GGG CAG CCC Gly Gin Pro CCC ATG CAG AAA Pro Met Gin Lys ATC TCC AAA GCC Ile Ser Lys Ala

AAA

Lys 125 CGA GAA Arg Glu 130 CCA CAG GTG TAC ACC CTG CCC CCA TCC Pro Gin Val Tyr Thr Leu Pro Pro Ser 135 GAT GAG CTG ACC Asp Glu Leu Thr AAG AAC CAG GTC AGC Lys Asn Gin Val Ser 145 ACC TGC CTG GTC Thr Cys Leu Val GGC TTC TAT CCC Gly Phe Tyr Pro CAC ATC GCC GTG GAG TGG GAG AGC AAT His Ile Ala Val Glu Trp Glu Ser Asn 165 CAG CCG GAG AAC Gin Pro Glu Asn AAC TAC Asn Tyr 175 i 1 WO 95/33059 PCT/US95/06530

AAG

Lys

AGC

Ser

TCA

Ser

AGC

Ser 225 (2)

ACC

Thr

AAG

Lys

TGC

Cys 210

CTC

Leu TCC GAC GGC TCC TTC TTC CTC TAC Ser Asp Gly Ser Phe Phe Leu Tyr 185 190 AGG TGG CAG CAG GGG AAC GTC TTC Arg Trp Gin Gin Gly Asn Val Phe 205 CTG CAC AAC CAC TAC ACG CAG AAG Leu His Asn His Tyr Thr Gin Lys 220

TGAACTAGT

INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 232 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Glu 1 Pro Lys Val Asp Tyr Asp Leu Arg Lys 145 (xi) SEQUENCE Pro Arg Ser Cys Glu Leu Leu Gly Asp Thr Leu Met Asp Val Ser His Gly Val Glu Val Asn Ser Thr Tyr Trp Leu Asn Gly 100 Pro Ala Pro Met 115 Glu Pro Gin Val 130 Asn Gin Val Ser DESCRIPTION: SEQ ID NO:4: Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Val Thr Glu Lys Ser Lys 105 Ile Pro Leu Leu Glu Lys Lys 75 Leu Lys Lys Ser Lys 155 Pro Val Val Gin Gin Ala Pro Thr Arg 160 His Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr c- i i t WO 95/33059 PCT/US95/06530 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 4171 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: huOSM-RBS (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 368..448 (ix) FEATURE: NAME/KEY: CDS LOCATION: 368..3307 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 449..3304 (xi) SEQUENCE DESCRIPTION: SEQ ID GGGCCGCCTC TGCACGTCCG CCCCGGAGCC CGCACCCGCG TCGGCCCGGC TCGTGGAGCC CTTCGCCCGC GGCGTGAGTA GCTCTGCTCG CGCCCTGCCG CTGCGCCGCC CTCGGTGGCT TGCTGTGCGG GAAAGAATCC GACAACTTCG CAGCCCATCC TGCAGCAGCC CGTTCCCCTC CTCGGTGCCG CCTCTGCCCA CCACCTGCGC TCGGACGGCG CTCGGAGGGT CCTCGCCCCC AGAACTG ATG GCT CTA TTT GCA GTC TTT CAG ACA Met Ala Leu Phe Ala Val Phe Gin Thr -27 -25 -20 CCCCACGCGC CGCCGAGGAC CCCCCGACCC GCCCGTCCCC TTTCCGACGG GCGAGCCCCG CGGCTGGACG CGACCGGGAG GCGTTTGCTT GGCTGGGCTA GGCCTGCCTA CCTGAAAACC ACA TTC TTC TTA ACA Thr Phe Phe Leu Thr e I WO 95/33059 PCT[US95/06530 TTG CTG TCC TTG AGG ACT TAC CAG Leu Leu Ser Leu Arg Thr Tyr Gin GAA GTC TTG GCT GAA CGT TTA Glu Val Leu Ala Glu Arg Leu 1 CCA TTG ACT CCT GTA Pro Leu Thr Pro Val TCA CTT Ser Leu AAA GTT TCC ACC Lys Val Ser Thr TCT ACG CGT CAG Ser Thr Arg Gin

AGT

Ser TTG CAC TTA CAA Leu His Leu Gin ACT GTC CAC AAC Thr Val His Asn CCT TAT CAT CAG Pro Tyr His Gin TTG AAA ATG GTA TTT CAG ATC CAG ATC AGT AGG ATT GAA ACA Leu Lys Met Val Phe Gin Ile Gin Ile Ser Arg Ile Giu Thr TCC AAT Ser Asn GTC ATC TGG Val Ile Trp CTG CAT TGG Leu His Trp GGG AAT TAC AGC Gly Asn Tyr Ser ACT GTG AAG TGG Thr Vai Lys Trp AAC CAG GTT Asn Gin Vai GCC ACA CAC Ala Thr His AGC TGG GAA TCT Ser Trp Giu Ser CTC CCT TTG GAA Leu Pro Leu Glu TTT GTA Phe Vai AGA ATA AAG AGT Arg Ile Lys Ser GTG GAC GAT GCC Vai Asp Asp Ala TTC CCT GAG CCA Phe Pro Giu Pro

AAT

Asn 100 TTC TGG AGC AAC Phe Trp Ser Asn AGT TCC TGG GAG GAA GTC AGT GTA CAA Ser Ser Trp Glu Giu Val Ser Vai Gin TCT ACT GGA CAG Ser Thr Giy Gin

GAT

Asp 120 ATA TTG TTC GTT Ile Leu Phe Val CCT AAA GAT AAG Pro Lys Asp Lys CTG GTG Leu Vai 130 GAA GAA GGC ACC AAT GTT ACC ATT Glu Giu Gly Thr Asn Val Thr Ile 135

TGT

Cys 140 TAC GTT TCT AGG Tyr Val Ser Arg AAC ATT CAA Asn Ile Gin 145 GGA GAA CAA Gly Glu Gin AAT AAT GTA Asn Asn Vai 150 TCC TGT TAT TTG Ser Cys Tyr Leu GGG APA CAG ATT Gly Lys Gin Ile CTT GAT Leu Asp 165 CCA CAT GTA ACT Pro His Val Thr

GCA

Ala 170 TTC AAC TTG AAT AGT GTG CCT TTC ATT Phe Asn Leu Asn Ser Val Pro Phe Ile

AGG

Arg 180 AAT AAA GGG ACA AAT ATC TAT TGT GAG Asn Lys Gly Thr Asn Ile Tyr Cys Glu AGT CAA GGA AAT Ser Gin Giy Asn 985 1033 1081 1129 AGT GAA GGC ATG Ser Giu Gly Met GGC ATC GTT CTT TTT GTC TCA AAA GTA Gly Ile Val Leu Phe Val Ser Lys Val 205 CTT GAG Leu Glu 210 GAG CCC AAG Glu Pro Lys

GAC

Asp 215 TTT TCT TGT GAA Phe Ser Cys Glu GAG GAC TTC AAG Glu Asp Phe Lys ACT rTG CAC Thr Leu His 225 WO 95/33059 PCTIUS95/06530 TGT ACT TGG GAT Cys Thr Trp Asp 230 CCT GGG ACG Pro Gly Thr ACT GCC TTG GGG Thr Ala Leu Gly TCT AAA CAA Ser Lys Gin CCT TCC Pro Ser 245 CAA AGC TAC ACT Gin Ser Tyr Thr

TTA

Leu 250 TTT GAA TCA TTT Phe Glu Ser Phe

TCT

Ser 255 GGG GAA AAG AAA Gly Glu Lys Lys

CTT

Leu 260 TGT ACA CAC AAA Cys Thr His Lys TGG TGT AAT TGG Trp Cys Asn Trp ATA ACT CAA GAC Ile Thr Gin Asp

TCA

Ser 275 CAA GAA ACC TAT Gin Glu Thr Tyr TTC ACA CTC ATA Phe Thr Leu Ile GAA AAT TAC TTA Glu Asn Tyr Leu AGG AAG Arg Lys 290 AGA AGT GTC Arg Ser Val AAT CCT TTT Asn Pro Phe 310 ATC CTT TTT AAC Ile Leu Phe Asn ACT CAT CGA GTT Thr His Arg Val TAT TTA ATG Tyr Leu Met 305 AAT GCC ATC Asn Ala Ile AGT GTC AAC TTT Ser Val Asn Phe

GAA

Glu 315 AAT GTA AAT GCC Asn Val Asn Ala

ACA

Thr 320 ATG ACC Met Thr 325 TGG AAG GTG CAC Trp Lys Val His

TCC

Ser 330 ATA AGG AAT AAT Ile Arg Asn Asn

TTC

Phe 335 ACA TAT TTG TGT Thr Tyr Leu Cys

CAG

Gin 340 ATT GAA CTC CAT Ile Glu Leu His GAA GGA AAA ATG ATG CAA TAC AAT GTT Glu Gly Lys Met Met Gin Tyr Asn Val 1177 1225 1273 1321 1369 1417 1465 1513 1561 1609 1657 1705 1753 1801 1849 ATC AAG GTG AAC Ile Lys Val Asn GAG TAC TTC TTA Glu Tyr Phe Leu GAA CTG GAA CCT Glu Leu Glu Pro GCC ACA Ala Thr 370 GAG TAC ATG Glu Tyr Met TGG AGT GAA Trp Ser Glu 390 CGA GTA CGG TGT Arg Val Arg Cys

GCT

Ala 380 GAT GCC AGC CAC Asp Ala Ser His TTC TGG AAA Phe Trp Lys 385 TGG AGT GGT CAG Trp Ser Gly Gin

AAC

Asn 395 TTC ACC ACA CTT GAA GCT GCT CCC Phe Thr Thr Leu Glu Ala Ala Pro TCA GAG Ser Glu 405 GCC CCT GAT GTC Ala Pro Asp Val AGA ATT GTG AGC TTG GAG CCA GGA AAT Arg Ile Val Ser Leu Glu Pro Gly Asn ACT GTG ACC TTA Thr Val Thr Leu TGG AAG CCA TTA Trp Lys Pro Leu AAA CTG CAT GCC AAT Lys Leu His Ala Asn GGA AAG ATC CTG Gly Lys Ile Leu

TTC

Phe 440 TAT AAT GTA GTT Tyr Asn Val Val

GTA

Val 445 GAA AAC CTA GAC Glu Asn Leu Asp AAA CCA Lys Pro 450 TCC AGT TCA GAG CTC CAT TCC ATT CCA GCA CCA GCC AAC Ser Ser Ser Glu Leu His Ser Ile Pro Ala Pro Ala Asn AGC ACA AAA Ser Thr Lys 465 WO 95/33059 PCT/US95/06530 CTA ATC CTT GAC Leu Ile Leu Asp 470 AGG TGT TCC Arg Cys Ser CAA ATC TGC GTC ATA GCC AAC AAC Gin Ile Cys Val Ile Ala Asn Asn 480 AGT GTG Ser Val 485 GGT GCT TCT CCT Gly Ala Ser Pro TCT GTA ATA GTC Ser Val Ile Val TCT GCA GAC CCC Ser Ala Asp Pro GAA AAC AAA GAG GTT GAG GAA GAA AGA ATT Glu Asn Lys Glu Val Glu Glu Glu Arg Ile

GCA

Ala 510 GGC ACA GAG GGT Gly Thr Glu Gly

GGA

Gly 515 TTC TCT CTG TCT Phe Ser Leu Ser AAA CCC CAA CCT Lys Pro Gin Pro GAT GTT ATA GGC Asp Val Ile Gly TAT GTT Tyr Val 530 GTG GAC TGG Val Asp Trp AAG AAT GTA Lys Asn Val 550 GAC CAT ACC CAG Asp His Thr Gin GTG CTC GGT GAT TTC CAG TGG Val Leu Gly Asp Phe Gin Trp 545 GGT CCC AAT ACC Gly Pro Asn Thr AGC ACA GTC ATT Ser Thr Val Ile ACA GAT GCT Thr Asp Ala TTT AGG Phe Arg 565 CCA GGA GTT CGA Pro Gly Val Arg

TAT

Tyr 570 GAC TTC AGA ATT Asp Phe Arg Ile TAT GGG TTA TCT ACA Tyr Gly Leu Ser Thr 575 GGA TAC TCT CAG GAA Gly Tyr Ser Gin Glu

AAA

Lys 580 AGG ATT GCT TGT TTA TTA GAG AAA AAA Arg Ile Ala Cys Leu Leu Glu Lys Lys

ACA

Thr 590 1897 1945 1993 2041 2089 2137 2185 2233 2281 2329 2377 2425 2473 2521 2569 CTT GCT CCT Leu Ala Pro CAC TCC TTC His Ser Phe GGT TTT ATA Gly Phe Ile 630 TCA GAC Ser Asp 600 AAC CCT CAC GTG CTG GTG GAT ACA TTG Asn Pro His Val Leu Val Asp Thr Leu 605 ACA TCC Thr Ser 610 CTG AGT TGG Leu Ser Trp AAA GAT Lys Asp 620 TAC TCT ACT GAA Tyr Ser Thr Glu TCT CAA CCT Ser Gin Pro 625 GCG AGG CAG Ala Arg Gin CAA GGG TAC CAT Gin Gly Tyr His TAT CTG AAA TCC Tyr Leu Lys Ser TGC CAC Cys His 645 CCA CGA TTT GAA Pro Arg Phe Glu GCA GTT CTT TCA Ala Val Leu Ser GGT TCA GAA TGT Gly Ser Glu Cys AAA TAC AAA ATT Lys Tyr Lys Ile AAC CCG GAA GAA Asn Pro Glu Glu GCA TTG ATT GTG Ala Leu Ile Val AAC CTA AAG CCA Asn Leu Lys Pro TCC TTC TAT GAG Ser Phe Tyr Glu TTC ATC ACT CCA Phe Ile Thr Pro TTC ACT Phe Thr 690 AGT GCT GGT Ser Ala Gly GGC CCC AGT GCT Gly Pro Ser Ala TTC ACG AAG GTC Phe Thr Lys Val ACG ACT CCG Thr Thr Pro 705 WO 95/33059 PCTUS95/06530 GAT GAA CAC Asp Giu His 710 TCC TCG ATG CTG Ser Ser Met Leu CAT ATC CTA CTG His Ile Leu Leu

CCC

Pro 720 ATG GTT TTC Met Vai Phe TGC GTC Cys Val 725 TTG CTC ATC ATG Leu Leu Ile Met ATG TGC TAC TTG Met Cys Tyr Leu AGT CAG TGG ATC Ser Gin Trp lie

AAG

Lys 740 GAG ACC TGT TAT Glu Thr Cys Tyr GAC ATC CCT GAC Asp Ile Pro Asp TAC AAG AGC AGC Tyr Lys Ser Ser CTG TCA TTA ATA Leu Ser Leu Ile

AAA

Lys 760 TTC AAG GAG AAC Phe Lys Giu Asn CAC CTA ATA ATA His Leu Ile Ile ATG AAT Met Asn 770 GTC AGT GAC Vai Ser Asp GGG ACA AAG Gly Thr Lys 790

TGT

Cys 775 ATC CCA GAT GCT ATT GAA GTT GTA AGC Ile Pro Asp Aia Ile Giu Vai Vai Ser AAG CCA GAA Lys Pro Glu 785 ACA GAA ACC Thr Giu Thr ATA CAG TTC CTA Ile Gin Phe Leu ACT AGG AAG TCA Thr Arg Lys Ser GAG TTG Glu Leu 805 ACT AAG CCT AAC Thr Lys Pro Asn TAC CTT Tyr Leu 810 TAT CTC CTT Tyr Leu Leu ACA GAA AAG AAT Thr Giu Lys Asn TCT GGC CCT GGC Ser Giy Pro Gly TGC ATC TGT TTT Cys Ile Cys Phe AAC TTG ACC TAT Asn Leu Thr Tyr 2617 2665 2713 2761 2809 2857 2905 2953 3001 3049 3097 3145 3193 3241 3289 CAG GCA GCT Gin Ala Ala TCT GAC Ser Asp 840 TCT GGC TCT TGT Ser Gly Ser Cys CAT GTT CCA GTA His Val Pro Val TCC CCA Ser Pro 850 AAA GCC CCA AGT ATG CTG GGA CTA Lys Ala Pro Ser Met Leu Giy Leu ACC TCA CCT GAA Thr Ser Pro Glu AAT GTA CTA Asn Val Leu 865 ATC CCA GCT lie Pro Ala AAG GCA OTA Lys Ala Leu 870 GAA AAA AAC TAC Glu Lys Asn Tyr AAC TCC CTG GGA Asn Ser Leu Gly GGA GAA Gly Glu 885 ACA AGT TTG AAT Thr Ser Leu Asn GTG TCC CAG TTG Val Ser Gin Leu TCA CCC ATG TTT Ser Pro Met Phe

GGA

Gly 900 GAC AAG GAC AGT Asp Lys Asp Ser CCA ACA AAC CCA Pro Thr Asn Pro GAG GCA CCA CAC Glu Ala Pro His TCA GAG TAT AAA Ser Glu Tyr Lys CAA ATG GCA GTC Gin Met Ala Val CTG CGT CTT GCC Leu Arg Leu Ala TTG CCT Leu Pro 930 CCC CCG ACC GAG AAT AGC AGC CTC TCC TCA ATT ACC CTT Pro Pro Thr Glu Asn Ser Ser Leu Ser Ser Ile Thr Len TTA GAT CCA Leu Asp Pro 945 ~ll~L~ j _i WO 95/33059 WO 9533059PCT/US95/06530 GGT GAA CAC TAC TGC TAACCAGCAT GCCGATTTCA TACCTTATGC TACACAGACA Gly Glu His Tyr Cys 950

TTAAGAAGAG,

CAATTTGCAG

CTATGGAACT

AGCATGCTTA

GACCTAAGGA

GTGGTCCTAG

TGAAGGCTTT

TTACTGAGCG

TACCTTGAAC

GGGCGGGGGG

ACAATTCTTC

TACTGTCAGT

ACCTCAGAAC

CAGAGCTGGC

GTCTGGTTT.A

TAACACTCCC

CCTTCTGCTG

TATGCATTAA

AAGTTCAGTT

AATAAAGGCC

TGTTTTATAA

GGATAAGAAT

CATAGCTCTA

TGTCAAGCTT

TGTACTTCTG

ATAAAAAGGA

ACCCTGTCAT

TATAAGACCA

CATTGGAGCA

TTTGTTCCAG

CCACTCTACA

TTTACTGAGG

ACAGGAGACA

AAAACTCACA

CTATAGTTCA

ATCTAATATA

ACTACAGTGA

CTCCATAGAC

ACGTATATCA

CACCAGTGGC

CTACAGTCTG

AGCTTGCCCT

GCTCACCTTT

GACTCCCACT

AAA.TATTTCC

TTAC,17hm~rC

GGTGTTTGAG

CTGACACAGT

TAAAATGTGT

AAGAATGGGA

ATCAGTATTC

CATAATTCCA

TGTTTTCACG

CTTGGTCCTT

GCTAGGTTAA

AGAGACGGCA

AGAACAGGAG

CAGTACTGTA

ATTAACAGCA

ATAGATTGTC

GCCAAAACAG

AAAATTAACT

GATGAATCAA

TTGGCAAGTA

TGCCATCATT

GTCACAGTTT

CGGCCGC

AATCCCAGTA

AGGCCAGAGG

GGATCATGGG

ACTTGAGCTT

CAGGGTGGCT

ATTATTATAT

AAATGTAAAT

ATTTTAGACT

CTGTGGGTGG

CAAGATTTCC

ACTTCTGACT

TTTGATGACT

TTGGTTCCTC

3344 3404 3464 3524 3584 3644 3704 3764 3824 3884 3944 4004 4064 4124 4171 TTTTCTTTCA AGAACTATAT ATAAATGACC INFORMATION FOR SEQ ID NO:6: 4 SEQUENCE CHARACTERISTICS: LENGTH: 979 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Ala Leu Phe Ala -25 DESCRIPTION: SEQ ID Val Phe Gln Thr Thr -20 NO: 6: Phe Phe Leu Met -27 Thr Leu Leu Ser Leu Thr Pro Arg Thr Tyr Val Ser Leu Gln Ser Glu Val Leu Ala Glu Arg Leu Pro Lys Val Ser Thr Asn 15 Val His Asn Leu Pro Ser Thr Arg Gln Ser Leu Leu Lys His Leu Gln Met Val Phe

'V)

Trp Val Gly Trp Thr Tyr His Gln Gln Ile Gln Ile Ser Arg Ile 45 Thr Val Lys Glu Thr Ser Asn Val Ile Asn Tyr Ser Trp Asn Gln Val Leu His WO 95/33059 WO 9533059PCTfUS95/06530 Trp Arg Trp, Gly Gly Val 150 Pro Lys Gly Lys Trp 230 Gin Thr Thr Val Phe 310 Trp Glu Val Met Ser Ie Ser Gin Thr 135 Ser His Gly Met Asp 215 Asp Ser His Tyr Asn 295 Ser Lys Leu Asn Ala 375 Trp Lys Asn Asp 120 Asn Cys Val Thr Lys 200 Phe Pro Tyr Lys Asn 280 Ile Val Val His Gly 360 Arg Giu Ser Trp 105 Ile Val Tyr Thr Asn 185 Gly Ser Gly Thr Asn 265 Phe Leu Asn His Gly 345 Giu Val Ser Leu Ser Leu Thr Leu Ala 170 Ile Ile Cys Thr Leu 250 Trp Thr Phe Phe Ser 330 Giu Tyr Arg Glu Leu 75 Val Asp Ser Trp Phe Val Ile Cys 140 Glu Gly 155 Phe Asn Tyr Cys Val Leu Glu Thr 220 Asp Thr 235 Phe Giu Cys Asn Leu Ile Asn Leu 300 Glu Asn 315 Ile Arg Gly Lys Phe Leu Cys Ala 380 Pro Asp Giu Phe 125 Tyr Lys Leu Giu Phe 205 Giu Ala Ser Trp Ala 285 Thr Val Asn Met Ser 365 Asp Leu Giu Ala Lys 95 Giu Val 110 Pro Lys Val Ser Gin Ile Asn Ser 175 Ala Ser 190 Val Ser Asp Phe Leu Gly Phe Ser 255 Gin Ile 270 Glu Asn His Arg Asn Ala Asn Phe 335 Met Gin 350 Glu Leu Ala Ser Cys Phe Ser Asp Arg His 160 Val Gin Lys Lys Trp 240 Gly Thr Tyr Val Thr 320 Thr Tyr Giu His Ala Thr Pro Giu Val Gin Lys Leu 130 Asn Ile 145 Gly Giu Pro Phe Gly Asn Vai Leu 210 Thr Leu 225 Ser Lys Giu Lys Gin Asp Leu Arg 290 Tyr Leu 305 Asn Ala Tyr Leu Asn Val Pro Aia 370 Phe Trp 385 Phe Asn 100 Ser Giu Asn Leu Arg 180 Ser Giu Cys Pro Leu 260 Gin Arg Asn Met Gin 340 Ile Giu Trp WO 95/33059 Glu Trp Ser 390 Ala Pro Asp Val Thr Leu Ile Le~a Phe ,440I Ser Glu Leu 455 Leu Asp Arg 470 Gly Ala Ser Lys Glu Val Leu Ser Trp 520 Trp Cys Asp 535 Val Gly Pro 550 Pro Gly Val Ile Ala Cys Pro Ser Asp 600 Phe Thr Leu 615 Ile Gin Gly 630 Pro Arg Phe Tyr Lys Ile Lys Pro Glu 680 Gly Val Phe 42£S a-yr His Cys Pro Glu 505 Lys His Asn Arg Leu 585 Asn Ser Tyr Giu Asp 665 Ser Gin Trp 410 Trp Asn Ser Ser Ala 490 Glu Pro Thr Thr Tyr 570 Leu Pro Trp His Lys 650 Asn Phe Thr Val Leu Vai 445 Aia Ile Ile Ile Gly 525 Val Thr Arg Lys Leu 605 Tyr Leu Leu Giu Phe 685 PCTIUJS95/06530 Ser Giu 405 His Thr 420 Gly Lys Ser Ser Leu Ile Ser Val 485 Giu Asn 500 Phe Ser Val Asp Lys Asn Phe Arg 565 Lys Arg 580 Leu Ala His Ser Gly Phe Cys His 645 Cys Lys 660 Asn Leu Ser Ala Gly Glu 695 Gly Pro Ser Ala Thr Phe Thr Lys Val Thr Thr Pro Asp Glu WO 95/33059 PCT/US95/06530 His Ser Ser Met Leu Ile His Ile Leu Leu Pro Met Val Phe Cys Val 710 715 720 725 Leu Leu Ile Met Val Met Cys Tyr Leu Lys Ser Gin Trp Ile Lys Glu 730 735 740 Thr Cys Tyr Pro Asp Ile Pro Asp Pro Tyr Lys Ser Ser Ile Leu Ser 745 750 755 Leu Ile Lys Phe Lys Glu Asn Pro His Leu Ile Ile Met Asn Val Ser 760 765 770 Asp Cys Ile Pro Asp Ala Ile Glu Val Val Ser Lys Pro Glu Gly Thr 775 780 785 Lys Ile Gin Phe Leu Gly Thr Arg Lys Ser Leu Thr Glu Thr Glu Leu 790 795 800 805 Thr Lys Pro Asn Tyr Leu Tyr Leu Leu Pro Thr Glu Lys Asn His Ser 810 815 820 S Gly Pro Gly Pro Cys Ile Cys Phe Glu Asn Leu Thr Tyr Asn Gln Ala 825 830 835 SAla Ser Asp Ser Gly Ser Cys Gly His Val Pro Val Ser Pro Lys Ala 840 845 850 Pro Ser Met Leu Gly Leu Met Thr Ser Pro Glu Asn Val Leu Lys Ala 855 860 865 Leu Glu Lys Asn Tyr Met Asn Ser Leu Gly Glu Ile Pro Ala Gly Glu 870 875 880 885 Thr Ser Leu Asn Tyr Val Ser Gin Leu Ala Ser Pro Met Phe Gly Asp 890 895 900 Lys Asp Ser Leu Pro Thr Asn Pro Val Glu Ala Pro His Cys Ser Glu 905 910 915 Tyr Lys Met Gin Met Ala Val Ser Leu Arg Leu Ala Leu Pro Pro Pro 920 925 930 Thr Glu Asn Ser Ser Leu Ser Ser Ile Thr Leu Leu Asp Pro Gly Glu 935 940 945 His Tyr Cys 950 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO 51 c- WO 95/33059 PCT/US95/06530 (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: FLAG peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asp Tyr Lys Asp Asp Asp Asp Lys 1 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: f LENGTH: 4 amino acids S(B) TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: spacer peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Asn Arg Tyr Val 1 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Phe Arg Xaa Arg Cys 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear I 3 I- i L WO 95/33059 PCTIUS95/06530 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Gln Ile Arg Cys 1 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Trp Ser Xaa Trp Ser 1 53 53

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Claims (25)

1. A purified receptor capable of binding oncostatin M, comprising a polypeptide and an oncostatin M receptor B-chain (OSM-RB) polypeptide, wherein: a) said gpl30 polypeptide is selected from the group consisting of: i) the gp130 polypeptide of SEQ ID NO:2; ii) a biologically active fragment of the polypeptide of and iii) a biologically active gpl30 polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence presented as amino acids 1 to 686 of SEQ ID NO:2; I b) said OSM-RB polypeptide is selected from the group consisting of: S iv) the OSM-RB polypeptide of SEQ ID NO:6; c v) a biologically active fragment of the polypeptide of and vi) a biologically active OSM-RB polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence presented as amino I acids 1 to 952 of SEQ ID NO:6.

2. A receptor according to claim 1, wherein said receptor comprises a soluble gp130 polypeptide covalently linked to a soluble OSM-RB polypeptide. i 3. A receptor according to claim 1 wherein said receptor comprises Scovalently linked to OSM-RB via a peptide linker. |i i c ne- o C\ c Io 1

4. A receptor according to -elaim 3, wherein said receptor is a recombinant fusion protein of the formula: R 1 -L-R 2 or R 2 -L-R 1 Swherein R 1 represents a soluble gpl30; R 2 represents a soluble OSM-RB, and L represents a peptide linker. A receptor according to claim 4, wherein said soluble gpl30 comprises amino acids 1 to y of SEQ ID NO:2, wherein y represents an integer between 308 and 597, inclusive; and said soluble OSM-RB comprises amino acids 1 to x of SEQ ID NO:6, wherein x is an integer between 432 and 714, inclusive. ac-o C

6. An isolated DNA sequence encoding o. receptor claim 4 or

7. A recombinant expression vector comprising o. DNA sequence claim 6.

8. A process for preparing a receptor according to claim 4 or 5, comprising culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes said fusion protein under conditions that promote expression of said fusion protein, and recovering said fusion protein.

9. A receptor according to claim 2 comprising a first fusion protein that I I V comprises an antibody Fc region polypeptide attached to the C-terminus of a soluble S° and a second fusion protein that comprises an antibody Fc region polypeptide attached to the C-terminus of a soluble OSM-RB, wherein said first fusion protein is linked to said Ssecond fusion protein via disulfide bonds between the Fc region polypeptides. A process for preparing a receptor according to claim 9, comprising culturing a host cell co-transfected with a first expression vector encoding said first fusion protein and with a second expression vector encoding said second fusion protein under conditions that promote expression of said first and second fusion proteins, and recovering said receptor.

11. A receptor according to claim 9, wherein said soluble gpl30 comprises amino acids 1 to y of SEQ ID NO:2, wherein y represents an integer between 308 and 597, inclusive; and said soluble OSM-R comprises amino acids 1 to x of SEQ ID NO:6, wherein x is an integer between 432 and 714, inclusive.

12. A receptor according to claim 1, wherein: said gpl30 polypeptide is a biologically active gpl30 polypeptide encoded by a DNA selected from the group consisting of: a) DNA comprising the coding region of the nucleotide sequence presented in SEQ ID NO:1; b) DNA capable of hybridizing to the DNA of under highly stringent conditions that include hybridization at 68 0 C followed by washing in 0.1X SSC/0.1% SDS at 63- 68 0 C; and i 56 c) DNA that encodes the amino acid sequence presented in SEQ ID NO:2; said OSM-RB polypeptide is a biologically active polypeptide encoded by a DNA selected from the group consisting of: DNA comprising the coding region of the nucleotide sequence presented in SEQ ID DNA capable of hybridizing to the DNA of under highly stringent conditions that include hybridization at 68 0 C followed by washing in 0.1X SSC/0.1% SDS at 63-68 0 C; and DNA that encodes the amino acid sequence presented in SEQ ID NO:6.

13. A pharmaceutical composition comprising a receptor according to any of claims 1 to 5, 9, 11, or 12, and a physiologically acceptable diluent or carrier.

14. An isolated DNA encoding an OSM-RP polypeptide, wherein said DNA is selected from the group consisting of: a) DNA encoding an OSM-R polypeptide comprising amino acids -27 to 952 of SEQ ID NO:6; b) DNA encoding an OSM-RB polypeptide comprising amino acids 1 to 952 of SEQ ID NO:6; c) DNA encoding a biologically active fragment of the polypeptide of and d) DNA encoding a biologically active OSM-RP polypeptide comprising an ,amino acid sequence that is at least 80% identical to the sequence presented as amino acids 1 to 952 of SEQ ID NO:6. 4i44

15. An isolated DNA according to claim 14, wherein said OSM-RB comprises 1 an amino acid sequence selected from the group consisting of amiiio acids -27 to 952 and amino acids 1 to 952 of SEQ ID NO:6. a

16. An isolated DNA according to claim 14, wherein said DNA encodes a soluble OSM-RB polypeptide.

17. An isolated DNA according to claim 16, where said soluble OSM-RB polypeptide comprises amino acids -27 to x or 1 to x of SEQ ID NO:6, wherein x is an integer between 432 and 714, inclusive.

18. An expression vector comprising a DNA according to any of claims 14 to 17.

19. A process for preparing an OSM-RB polypeptide, comprising culturing a host cell transformed with a vector according to claim 18 under conditions promoting expression of OSM-RB, and recovering the OSM-RB polypeptide. An OSM-RB polypeptide encoded by a DNA according to any of claims 14 to 17.

21. An OSM-RB polypeptide according to claim 20, wherein said OSM-RB is a soluble OSM-RB polypeptide. 0 0 0

22. A soluble OSM-RB according to claim 21, comprising amino acids -27 to x o or 1 to x of SEQ ID NO:6, wherein x is an integer between 432 and 714, inclusive. 0 00 °23. A purified OSM-RB polypeptide selected from the group consisting of: a) the OSM-RB polypeptide of SEQ ID NO:6; b) a biologically active fragment of the polypeptide of and c) a biologically active OSM-RB polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence presented as amino acids 1 to 952 of SEQ ID NO:6.

24. An OSM-RB polypeptide according to claim 23, wherein said OSM-RB is encoded by the OSM-RB cDNA in the recombinant vector deposited in strain ATCC 69675. 4 6 An OSM-RB polypeptide according to claim 23, comprising amino acids 1 to 952 of SEQ ID NO:6.

26. A fusion protein comprising a soluble OSM-R3 polypeptide according to claim 21 and an antibody Fc polypeptide.

27. An antibody that is immunoreactive with an OSM-RB polypeptide according to claim 23.

28. An antibody according to claim 27, wherein said antibody is a monoclonal antibody that is immunoreactive with the OSM-RB polypeptide of SEQ ID NO:6. r 1 *I 9" 58

29. The use of a receptor according to any of claims 1 to 5, 9, 11, or 12, for binding oncostatin M. The use of a receptor according to any of claims 1 to 5, 9, 11, or 12, for inhibiting a biological activity mediated by oncostatin M.

31. The use of a receptor according to any of claims 1 to 5, 9, 11 or 12, in the preparation of a medicament for treating a disorder mediated by oncostatin M.

32. A receptor according to claim 1 substantially as hereinbefore described with reference to any one of the examples. DATED: 20 May, 1997 PHILLIPS ORMONDE FITZPATRICK Attorneys for: IMMUNEX CORPORATION CWINWORDACIENODELETEXSP26016.DOC t i it t4k, J